Introduction Bioo Scientific

Transcription

1 Next Generation Sequencing Catalog

2 Introduction Bioo Scientific Bioo Scientific is a global life science company headquartered in Austin, TX, committed to providing innovative products and superior customer service. We redefine the limits of what s possible in Next Generation Sequencing, affording unprecedented throughput and efficiency for faster, higher-quality results. We are continually adding to our product line to increase the sensitivity, flexibility and speed of NGS experiments, driving breakthroughs in the field of genomics. Quality and delivery time are important considerations when deciding upon a vendor for your library preparations kits and reagents. Our focus has been developing enzymes (ligases and polymerases) that deliver the highest efficiency to achieve robust results while minimizing library prep associated bias. We ve designed our flagship library preparation solutions to optimize adapter ligation efficiency, generating a greater number of unique reads, higher sequence diversity, and greater coverage. Bioo Scientific also has an optimized inventory management system to ensure overnight delivery. In recognition of our achievements, Bioo Scientific has also been honored to receive the following awards: 2013 Greater Austin Business Technology Award 2013 APEX Award for Publication Excellence Print Ads & Advertorials 2011 Greater Austin Business Export Award 2009 Tech Innovator of the Year Award Biotech/Medical Division 2

3 Table of Contents DNA-Seq Library Prep - Illumina Compatible 04 Table of Contents DNA-Seq Library Prep - Ion PGM & Proton Compatible 28 DNA-Seq Library Prep - SOLiD Compatible 32 RNA-Seq Library Prep 38 Next-Gen Sequencing Automation 52 DNA Fragmentation & Nucleic Acid Isolation 56 Ordering Instructions 62 3

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5 DNA-Seq Library Prep Illumina Compatible 5

6 For automated solutions see page 54. DNA-Seq Library Prep - Illumina Compatible NEXTflex Rapid DNA Sequencing Kits for Illumina Flexible amounts of input DNA from 1 ng to 1 µg Fast workflow requiring 2 hours or less, with minimal hands-on time Enhanced Adapter Ligation Technology offers a larger number of unique sequencing reads Creates libraries with less bias than alternative methods Automation-friendly workflow is compatible with liquid handlers Flexible barcode options Up to 96 unique barcodes Compatible with Illumina HiSeq, MiSeq and GAIIx platforms Prepare single, paired-end and multiplexed genomic DNA libraries Bulk pricing available to meet research goals Bioo Scientific s NEXTflex Rapid DNA-Seq Kit allows researchers to build sequencing-ready libraries for sequencing on Illumina sequencing platforms, from as little as 1 ng of DNA, in two hours or less. This kit is ideal for use with genomic DNA, FFPE samples, ChIP DNA and low input clinical samples. The NEXTflex Rapid DNA-Seq Kit is highly flexible in terms of sample requirements, accommodating a 3-log range of input DNA amounts from 1 ng to 1 µg. A bead-based, gel-free size selection protocol eliminates the need for agarose gels in size selection, and the availability of up to 96 unique adapter barcodes facilitates high-throughput applications. The NEXTflex Rapid DNA-Seq Kit incorporates Enhanced Adapter Ligation Technology, which facilitates ligation of long adapters, resulting in longer and more diverse sequencing reads. Bioo Scientific s NEXTflex Ligation and Polymerase reaction mixes ensure the highest quality libraries for superior performance. For multiplexing, The NEXTflex Adapters are long, annealed adapters containing indexed sequences that offer an improved multiplexing workflow and flexible setup. These barcodes can be used with single, paired-end and multiplex reads. The NEXTflex Rapid DNA-Seq Kit is designed to be used with the NEXTflex and NEXTflex-96 DNA Barcodes for 10 ng or more of starting material or the NEXTflex and NEXTflex-96 ChIP- Seq Barcodes for less than 10 ng of starting material. These barcodes are available in sets of 6, 12, 24, 48 and 96 unique indexed adapters. Figure 1. The NEXTflex Rapid DNA-Seq Kit features a streamlined library prep protocol to help you meet your research goals faster than you thought possible. End Polishing Adapter Ligation PCR 1 ng 1 µg 2 hours or less Figure 2. Higher yield obtained by NEXTflex Rapid DNA-Seq Kit compared to competitor kit. The final nanomolar concentration measurements of each triplicate PCR condition by qpcr. Each triplicate Ct measurement was averaged, converted to pm (then nm) using the standard curve, determined, then plotted with standard deviation against the other PCR conditions NEXTflex, 6 cycles Phusion, 6 cycles NEXTlfex, 8 cycles Phusion, 8 cycles Library Samples (nm) STDEV Concentration (pm) NEXTflex, 6 cycles Phusion, 6 cycles NEXTflex, 8 cycles Phusion, 8 cycles Library Samples 6

7 Competitive Analysis The NEXTflex Rapid DNA-Seq Kit is compared below with Competitor N s Kit, illustrating the advantage of Bioo Scientific s Enhanced Adapter Ligation Technology. The NEXTflex Rapid DNA-Seq Kit and the competitor kit were used to prepare samples through ligation. All samples were then purified and size-selected using an identical AMPureXP bead cleanup step. Triplicate overlays of the three ligation product peaks for NEXTflex Rapid DNA-Seq and Competitor N s kits were generated. Integration of the ligation product peaks using the Agilent 2100 Bioanalyzer software produces size-independent values for percentage of product comprising each peak. These values were averaged for the triplicate ligation reactions for each sample. The resulting comparison of unligated, singly-ligated, and doubly-ligated products was graphed. % Representation Non-ligated Singly-ligated Doubly-ligated Sample Peak 1 Peak 2 Peak 3 NEXTflex Rapid DNA-Seq Competitor N s Kit Figure 3. Overlays of the triplicate samples of NEXTflex Rapid DNA-Seq (left) and Competitor N s kit (right). Figure 5. Average triplicate values for the desired doubly-ligated products for NEXTflex Rapid DNA- Seq and Competitor N prepared libaries. The NEXTflex libraries produced 47.1% relative conversion compared to Competitor N s 21.5%. DNA-Seq Library Prep - Illumina Compatible Figure 4. Direct comparison of NEXTflex Rapid DNA-Seq to Competitor N s kit by analysis of proportions of ligation products. Red portions of the graph indicate non-ligated product proportion; yellow, singly-ligated; green, the desired doubly-ligated amplicon NEXTflex Rapid DNA-Seq Competitor N s Kit 0.0 NEXTflex Rapid DNA-Seq Competitor N s Kit NEXTflex Rapid DNA Sequencing Kit 8 rxns NEXTflex Rapid DNA Sequencing Kit 48 rxns NEXTflex ChIP-Seq Barcodes rxns NEXTflex ChIP-Seq Barcodes rxns NEXTflex ChIP-Seq Barcodes rxns NEXTflex ChIP-Seq Barcodes rxns NEXTflex-96 ChIP-Seq Barcodes rxns NEXTflex DNA Barcodes rxns NEXTflex DNA Barcodes rxns NEXTflex DNA Barcodes rxns NEXTflex DNA Barcodes rxns NEXTflex-96 DNA Barcodes rxns 7

8 For automated solutions see page 54. DNA-Seq Library Prep - Illumina Compatible NEXTflex DNA Sequencing Kits for Illumina Enhanced Adapter Ligation Technology offers a larger number of unique sequencing reads Completely gel-free library prep protocol available Flexible barcode options - Kits contain 6, 12, 24, 48 or 96 unique barcodes Cost-effective library prep solution Simplified workflow with master mixes reduces hands-on time Compatible with targeted sequence capture Bead-based cleanup Compatible with the Illumina GAIIx, HiSeq and MiSeq sequencing platforms The NEXTflex DNA Sequencing Kit is designed to prepare single, paired-end and multiplexed genomic DNA libraries for sequencing using Illumina GAIIx, HiSeq and MiSeq platforms. The NEXTflex DNA Sequencing Kit features Enhanced Adapter Ligation Technology, resulting in library preps with a larger number of unique sequencing reads. With an improved enzymatic ligation mix, ligations can now be performed with longer adapters and higher binding efficiencies. This NEXTflex kit simplifies workflow by using master mixed reagents and magnetic bead based cleanup, reducing pipetting and eliminating time consuming steps in library preparation. In addition, the availability of up to 96 unique adapter barcodes makes the NEXTflex kits the most high-throughput kits available (see page 26 for more information about the NEXTflex DNA Barcodes). What is Enhanced Adapter Ligation Technology? By using ligases that are designed to repair DNA nicks at a significantly higher rate (>10 9 compared to 10 6 ) than traditional commercial T4 DNA ligases, Bioo Scientific has revolutionized adapter ligation in library preparation. This, along with proprietary modifications to the adapter sequences designed to reduce dimer formation and increase dual adapter ligation, comprise Enhanced Adapter Ligation Technology. Using NEXTflex ligation mixes, the user can expect a significantly greater number of unique reads, sequence diversity and coverage. Sequencing-Ready DNA Libraries from 1 ng 1 µg of DNA in 2 Hours or Less < 2 hour library construction Largest number of sequencing reads available Broad input range Visit Page 6 of the catalog or to accelerate your library prep. 8

9 NEXTflex DNA Sequencing Kit Protocol GENOMIC DNA FRAGMENT END-REPAIR ADD A = N = A = T = Adapters with Cluster Sequence DNA-Seq Library Prep - Illumina Compatible ADD ADAPTERS LIGATION GEL FREE SPRI SIZE SELECTION PCR GEL SIZE SELECTION BEAD CLEANUP BRIDGE AMPLIFICATION (CLUSTER GENERATION) COMPLEXITY REDUCTION (SEQUENCE CAPTURE) 9

10 DNA-Seq Library Prep - Illumina Compatible New, Completely Gel-Free, DNA Library Prep Protocol Bioo Scientific has updated the NEXTflex DNA Sequencing Kit to include an optional protocol which allows you to completely eliminate the gel purification step. The alternative bead-based protocol reduces the required sample prep time by two hours and allows for complete automation of DNA library prep. Deep Sequencing of the E. coli Genome As can be seen in Table 1, sequencing of the E. coli genome was performed using the NEXTflex DNA Sequencing Kit and Barcodes and compared to Company X s protocol. The NEXTflex Enhanced Adapter Ligation Technology resulted in significantly improved reads, coverage and assembly. The E. coli genome was assembled with fewer scaffolds (result of connecting contigs by linking data), had an increased average maximum scaffold size and N50. Table 1. Summary of Sequence Data between NEXTflex and Competitor X s Libraries NEXTflex DNA Sequencing Kit Competitor X s Protocol Sample E. coli E. coli Genome Size 5 Mb 5 Mb Read Length 36 bp 36 bp Insert Size (bp) Number of Reads 29,718,673 19,311,940 Read Coverage Perfectly Mapped Reads 96% 89% # of Unknown Bases 3,201 6,540 Contig N50 102,408 95, NEXTflex DNA Sequencing Kit 8 rxns NEXTflex DNA Sequencing Kit 48 rxns NEXTflex DNA Barcodes rxns NEXTflex DNA Barcodes rxns NEXTflex DNA Barcodes rxns NEXTflex DNA Barcodes rxns NEXTflex-96 DNA Barcodes (in 96-well plate format) 768 rxns NEXTflex-96 DNA Barcodes (in tubes) 768 rxns 10

11 NEXTflex ChIP Sequencing Kits for Illumina Low 1-10 ng input requirement For use with ChIP or genomic DNA samples Faster protocol - Bead-based cleanup shaves hours off of library prep Enhanced Adapter Ligation Technology offers a larger number of unique sequencing reads 96 barcoded adapters for multiplexing contain embedded index sequence Automation-friendly workflow is compatible with liquid handlers Flexible barcode options Kits contain 6, 12, 24, 48 or 96 unique barcodes Compatible with Illumina GAIIx, HiSeq and MiSeq sequencing platforms The NEXTflex ChIP-Seq Kit contains specially designed enzymes and buffers needed for library preparation of chromatin immunoprecipitated (ChIP) DNA or small genomic DNA samples for next generation sequencing. The NEXTflex ChIP-Seq Kit features Bioo Scientific s proprietary Enhanced Adapter Ligation Technology (see page 8 for more information) resulting in library preps with a larger number of unique sequencing reads. With an improved enzymatic ligation mix, ligations can now be performed with longer adapters and higher binding efficiencies. The NEXTflex ChIP-Seq Kit simplifies workflow by using master mixed reagents and magnetic bead based cleanup, reducing pipetting and eliminating time consuming steps in library preparation. Using NEXTflex ChIP-Seq Barcodes (see page 26 for more information) with Enhanced Adapter Ligation Technology, the user can index up to 96 samples at once, providing an unparalleled ChIP sequencing capacity. DNA-Seq Library Prep - Illumina Compatible NEXTflex ChIP Sequencing Kit 8 rxns NEXTflex ChIP Sequencing Kit 48 rxns NEXTflex ChIP Sequencing Barcodes rxns NEXTflex ChIP Sequencing Barcodes rxns NEXTflex ChIP Sequencing Barcodes rxns NEXTflex ChIP Sequencing Barcodes rxns NEXTflex ChIP Sequencing Barcodes rxns Sequencing-Ready DNA Libraries from 1 ng 1 µg of DNA in 2 Hours or Less < 2 hour library construction Largest number of sequencing reads available Broad input range Low input requirements ideal for ChIP-Seq experiments Visit Page 6 of the catalog or to accelerate your library prep. 11

12 DNA-Seq Library Prep - Illumina Compatible NEXTflex ChIP Sequencing Kit Protocol IMMUNOPRECIPITATION END-REPAIR PROTEIN = N = A = T = Adapters with Cluster Sequence 30 Minutes (Optional Stop Point) ADD A 30 Minutes ADD ADAPTERS LIGATION 15 Minutes (Optional Stop Point) PRE-SIZE SELECTION PCR / SIZE SELECTION 1-2 Hours (Optional Stop Point) PCR 1 Hour (Optional Stop Point) BEAD CLEANUP BRIDGE AMPLIFICATION (CLUSTER GENERATION) COMPLEXITY REDUCTION (SEQUENCE CAPTURE) 12

13 NEXTflex Methyl-Seq 1 Kits for Illumina Methylome-level assessment with broad genome coverage Make methyl rich libraries using methylated DNA immunoprecipitation (MeDIP) or MBD capture (MeCAP) Quantify absolute DNA methylation levels Identify differentially methylated regions (DMRs) Enhanced Adapter Ligation Technology with NEXTflex Ligation Flexible barcode options 6, 12, 24, 48 or 96 unique indices Gel-free and bead-based cleanup protocols Automation-friendly workflow is compatible with liquid handlers Barcoded adapters for multiplexing contain embedded index sequence Functionally validated with Illumina MiSeq, GAIIx and HiSeq platforms The NEXTflex Methyl-Seq 1 Kit is designed to enrich and prepare single, paired-end and multiplexed methylated DNA libraries for sequencing using Illumina MiSeq, GAIIx and HiSeq platforms. NEXTflex Methyl-Seq utilizes a versatile immune-capture (MeDIP) or methyl-cpg-binding domain (MeCAP) method for detection of methylated DNA, allowing the user to easily assess the methylation state of the genome. DNA-Seq Library Prep - Illumina Compatible This kit features Enhanced Adapter Ligation Technology (see page 8 for more information), resulting in library preps with a larger number of unique sequencing reads. This specially designed NEXTflex ligation enzymatic mix allows users to perform ligations with longer adapters and better ligation efficiencies. The NEXTflex Methyl-Seq 1 Kit simplifies workflow by using master mixed reagents and magnetic bead based cleanup, reducing pipetting and eliminating time consuming steps in library preparation. In addition, the availability of the NEXTflex DNA Barcodes (see pg 26) with up to 96 unique adapter indices makes the NEXTflex kits the most high-throughput kits available. Table 2: Sequence Comparison between Differentiated and Non-differentiated Cells Sample Culture Passage PCR Cycles Capture Method Format Unique Mapped Reads DMRs Covered mcs Covered H MeDIP Single Read 120,154, % 91.38% HDF - 9 MeDIP Paired Read 79,286, % 93.49% HDF - 9 MeCAP Paired Read 76,884, % 94.50% HDF-iPSC 15 8 MeCAP Paired Read 83,290, % 87.64% Figure 6. DNA Methylome, Genome Scale HDF-Fibroblast HDF H1 HDF-iPSC Chromosome 10: 131,130, ,030,027 mc(atc)g mc(atc)(atc) Figure 6 shows that on a genome scale, DNA methylomes of human embryonic stem cells and ipscs transformed from a human dermal fibroblast cell are similar to one another and highly distinct from the primary somatic cell lines. 13

14 DNA-Seq Library Prep - Illumina Compatible Figure 7. Frequency of DNA Methylation at Both CG and Non-CG sites Suggest ipscs Resemble ES and are Distinct from Somatic Cells Methylated CG/CG (%) HDF-Fibroblast Figure 7 shows that high sequence coverage of four DNA methylomes allowed interrogation of 87-94% of genomic methylated cytosines using both MeDIP and MeCAP enrichment. Pluripotent cell coverage is significantly different from somatic cell lines using both enrichment methods. HDF HDF-iPSC H NEXTflex Methyl Sequencing 1 Kit 8 rxns NEXTflex Methyl Sequencing 1 Kit 48 rxns NEXTflex DNA Barcodes rxns NEXTflex DNA Barcodes rxns NEXTflex DNA Barcodes rxns NEXTflex DNA Barcodes rxns NEXTflex-96 DNA Barcodes - 96 (in 96-well plate format) 768 rxns NEXTflex-96 DNA Barcodes - 96 (in tubes) 768 rxns 14

15 NEXTflex Methyl-Seq 1 Kit Protocol GENOMIC DNA FRAGMENT END REPAIR 30 Minutes DNA-Seq Library Prep - Illumina Compatible ADENYLATION 30 Minutes LIGATION 15 Minutes METHYLATED DNA ENRICHMENT PRE-SIZE SELECTION PCR 15 Minutes GEL SIZE SELECTION 1-2 Hours PCR 1 Hour BRIDGE AMPLIFICATION (CLUSTER GENERATION) COMPLEXITY REDUCTION (SEQUENCE CAPTURE) 15

16 DNA-Seq Library Prep - Illumina Compatible NEXTflex Bisulfite-Seq Kits for Illumina Compatible with total bisulfite-seq and reduced representation Single nucleotide resolution of methylation sites Uracil insensitive polymerase designed for bisulfite-converted DNA Methylome-level assessment with broad genome coverage Enhanced Adapter Ligation Technology with NEXTflex Ligation Bead-based cleanup Automation-friendly workflow is compatible with liquid handlers Methylated barcoded adapters for multiplexing contain embedded index sequence Functionally validated with Illumina MiSeq, GAIIx and HiSeq platforms The NEXTflex Bisulfite-Seq Kit is designed to work with reduced representation or total bisulfite-converted DNA libraries and is compatible with single, paired-end and multiplexed DNA libraries on Illumina MiSeq, GAIIx and HiSeq platforms. NEXTflex Bisulfite-Seq with reduced representation utilizes a restriction enzyme that leads to selective amplification of CpG regions, resulting in focused sequence depth. The NEXTflex Msp1 Restriction Enzyme, which is available separately, has been optimized for reduced representation studies when used in conjunction with the NEXTflex Bisulfite-Seq Kit. The NEXTflex Bisulfite-Seq Kit can also be used without the Msp1 Restriction Enzyme for genome-wide methylseq analysis, including under-represented CpG regions. This kit features Enhanced Adapter Ligation Technology, resulting in library preps with a larger number of unique sequencing reads. This specially designed NEXTflex ligation enzymatic mix allows users to perform ligations with longer adapters and better ligation efficiencies. The NEXTflex Bisulfite-Seq Kit is a versatile kit designed to facilitate assessment of the methylation state of the genome and simplify workflow by using master mixed reagents and magnetic bead based cleanup, reducing pipetting and eliminating time-consuming steps in library preparation. In addition, the availability of the methylated NEXTflex Bisulfite-Seq barcoded adapters make multiplexing possible NEXTflex Bisulfite Sequencing Kit 8 rxns NEXTflex Bisulfite Sequencing Kit 48 rxns NEXTflex Bisulfite Sequencing Barcodes rxns NEXTflex Bisulfite Sequencing Barcodes rxns NEXTflex Msp1 Restriction Enzyme 8 rxns NEXTflex Msp1 Restriction Enzyme 48 rxns 16

17 NEXTflex Bisulfite-Seq Kit Protocol GENOMIC DNA FRAGMENT END-REPAIR = N = A = T = Adapters with Cluster Sequence 30 Minutes (Optional Stop Point) DNA-Seq Library Prep - Illumina Compatible ADD A 30 Minutes ADD METHYLATED ADAPTERS LIGATION OF METHYLATED ADAPTERS 15 Minutes (Optional Stop Point) SIZE SELECTION 1-2 Hours (Optional Stop Point) BISULFITE CONVERSION 3 Hours PCR 1 Hour (Optional Stop Point) BEAD CLEANUP BRIDGE AMPLIFICATION (CLUSTER GENERATION) COMPLEXITY REDUCTION (SEQUENCE CAPTURE) 17

18 DNA-Seq Library Prep - Illumina Compatible NEXTflex PCR-Free DNA Sequencing Kits for Illumina Eliminates amplification bias and poor sequence representation Improves read mapping Reduces duplicate sequences Better de novo assembly Completely gel-free protocol Significantly faster library preparation time The NEXTflex PCR-Free DNA Sequencing Kit offers all the advantages of the original NEXTflex DNA Sequencing Kit, in addition to completely eliminating the need for amplification. Amplifying AT or GC rich genomic regions often leads to sequence biased nucleotide compositions and poses a serious challenge during analysis. Using specially designed master-mixed enzymes, the NEXTflex PCR-Free DNA Sequencing kit completely eliminates the need for amplification, enabling better read mapping and a reduction in duplicate sequences, leading to reduced sequence cost and bias, for more representative base identities and better de novo assembly. In assessing bias, the presence of low quality and high GC reads often cannot be aligned against a reference genome. High GC-containing profiles tend to shift toward higher GC content, an indication of poor base representation. Eliminating PCR removes this content bias for more representative reads. PCR is also the cause of a significant number of gene duplicates, which increases sequence cost, adapter dimers and noise in cluster detection. Using the NEXTflex PCR-Free Kit, the number of duplicate sequences is reduced, ensuring a more representative matched number of reads. While the quantity of template generated using the NEXTflex PCR-Free DNA Sequencing Kit is lower than the NEXTflex DNA Sequencing Kit, library quantification by qpcr demonstrates that from as little as 0.5 µg of DNA, a sufficient amount of bp PCR-free library can be obtained for greater than 600 high density HiSeq lanes. The NEXTflex PCR-Free Barcodes, available in sets of up to 48 barcodes, can be used to multiplex these reactions (see page 26 for more information) NEXTflex PCR-Free DNA Sequencing Kit 8 rxns NEXTflex PCR-Free DNA Sequencing Kit 48 rxns NEXTflex PCR-Free DNA Barcodes rxns NEXTflex PCR-Free DNA Barcodes rxns NEXTflex PCR-Free DNA Barcodes rxns NEXTflex PCR-Free DNA Barcodes rxns 18

19 NEXTflex PCR-Free DNA Sequencing Kit Protocol GENOMIC DNA FRAGMENT END-REPAIR = N = A = T = Adapters with Cluster Sequence 30 Minutes (Optional Stop Point) DNA-Seq Library Prep - Illumina Compatible SIZE SELECTION 1 Hour ADD A 30 Minutes ADD ADAPTERS LIGATION 15 Minutes (Optional Stop Point) BEAD CLEANUP BRIDGE AMPLIFICATION (CLUSTER GENERATION) 19

20 DNA-Seq Library Prep - Illumina Compatible NEXTflex Pre-Capture Combo Kit (NimbleGen SeqCap EZ Library Compatible) for Illumina Complete Kit Library prep, barcodes and barcode blockers compatible with NimbleGen s SeqCap EZ capture systems Simplifies and reduces the cost of target capture Multiplex Compatible - 96 barcodes and barcode blockers available Optimized - Enhanced Adapter Ligation Technology offers larger number of unique reads Completely gel-free library prep protocol available Simplified workflow with master mixes that reduces hands on time Bead-based cleanup protocol Compatible with the Illumina GAIIx and HiSeq sequencing platforms The NEXTflex Pre-Capture Combo Kit (NimbleGen SeqCap EZ Library Compatible) is a complete library prep solution designed for library preparation, multiplexing and barcode blocking, upstream of Roche NimbleGen s SeqCap EZ capture kits. The NEXTflex Pre-Capture Combo Kit (NimbleGen SeqCap Compatible) is designed to prepare single, paired-end and multiplexed genomic DNA libraries for target capture and Illumina compatible sequencing. This kit features Enhanced Adapter Ligation Technology resulting in library preps with a larger number of unique sequencing reads. With Bioo Scientific s improved enzymatic ligation mix, ligations can be performed with longer adapters and higher binding efficiencies. The NEXTflex Pre-Capture Combo Kit simplifies workflow by using master mixed reagents and magnetic bead based cleanup, reducing pipetting and eliminating time consuming steps in library preparation. Table 3. Mapping and coverage statistics for twelve sample libraries demonstrates the efficacy of pre-capture multiplexing using NEXTflex library and blocking oligo reagents. Samples NEXTflex Index Targeted Bases Raw Reads Gb of Sequence % Raw Reads Mapped % Unique Reads Mapped % Mapped Reads on Target Unique Gb of Sequence on Target Average Coverage % Sensitivity in Detecting Known SNPs % Specificity in Detecting Known SNPs 1 CGATGT ,548, TGACCA ,218, ACAGTG ,026, GCCAAT ,589, CAGATC ,077, CTTGTA ,412, ATCACG ,317, TTAGGC ,994, ACTTGA ,344, CATCAG ,371, TAGCTT ,844, GGCTAC ,987, NEXTflex Pre-Capture Combo Kit - 6 Barcodes 48 rxns NEXTflex Pre-Capture Combo Kit - 12 Barcodes 96 rxns NEXTflex Pre-Capture Combo Kit - 24 Barcodes 192 rxns NEXTflex Pre-Capture Combo Kit - 96 Barcodes 768 rxns NEXTflex DNA Barcode Blockers - 6 for SeqCap EZ Library 48 rxns NEXTflex DNA Barcode Blockers - 12 for SeqCap EZ Library 96 rxns NEXTflex DNA Barcode Blockers - 24 for SeqCap EZ Library 192 rxns NEXTflex-96 DNA Barcode Blockers - 96 for SeqCap EZ Library 8 x 96 pooled libraries 20

21 NEXTflex Pre-Capture Combo Kit Protocol GENOMIC DNA FRAGMENT END REPAIR ADD A = N = A = T = Adapters with Cluster Sequence 30 Minutes (Optional Stop Point) 30 Minutes DNA-Seq Library Prep - Illumina Compatible ADD ADAPTERS LIGATION 15 Minutes (Optional Stop Point) SIZE-SELECTION 1-2 Hours (Optional Stop Point) PCR 1 Hour (Optional Stop Point) BEAD CLEANUP TARGET CAPTURE Using NimbleGen SeqCap EZ Library Kit SEQUENCING 21

22 DNA-Seq Library Prep - Illumina Compatible NEXTflex 16S V4 Amplicon-Seq Kit for Illumina Fast, two-step library prep protocol Flexible barcode options - Kits containing 4, 12, 24, 48, 96 or 288 unique barcodes Automation-friendly workflow is compatible with liquid handlers Cost-effective library prep solution Only 50 ng of input required Functionally validated with Illumina MiSeq and HiSeq sequencing platforms The NEXTflex 16S V4 Amplicon-Seq Kit is a validated amplicon-seq library prep kit developed to simplify bacterial metagenomics studies using Illumina MiSeq and HiSeq platforms. This kit allows users to go from sample to sequence in two hours, making it the fastest ampliconseq library prep kit available. The NEXTflex 16S V4 Amplicon-Seq Kit offers a fast two step, gel-free protocol that reduces library prep time to 2 hours and allows for complete automation of bacterial metagenomics studies. There are two main steps involved in the NEXTflex 16S V4 Amplicon-Seq Kit protocol: amplification and cleanup. Primers included in the NEXTflex 16S V4 Amplicon-Seq Kit are designed to hybridize to the fourth hypervariable (V4) domain of microbial 16S ribosomal RNA (rrna) genes allowing for amplification and sequencing of the variable regions. The single cleanup step ensures maximum recovery of amplicons for downstream sequencing. NEXTflex 16S V4 Amplicon-Seq Kit Protocol BACTERIAL GENOMIC DNA V4 Reverse V4 Forward PCR 2 Hours BEAD CLEANUP SEQUENCING NEXTflex 16S V4 Amplicon-Seq Kit - 4 Barcodes 16 rxns NEXTflex 16S V4 Amplicon-Seq Kit - 12 Barcodes 48 rxns NEXTflex 16S V4 Amplicon-Seq Kit - 24 Barcodes 96 rxns NEXTflex 16S V4 Amplicon-Seq Kit - 48 Barcodes 192 rxns NEXTflex 16S V4 Amplicon-Seq Kit - 96 Barcodes 384 rxns NEXTflex 16S V4 Amplicon-Seq Kit Barcodes 1,152 rxns 22

23 NEXTflex DNA Sequencing Kit (BioMek FXp Compatible) for Illumina Library preparation kit for use with the Biomek FXp Liquid Handler Optimized - Enhanced Adapter Ligation Technology offers larger number of unique reads Multiplex Compatible Sets of up to 96 barcodes are available Cost-effective library prep solution Automated bead-based cleanup protocol Compatible with Illumina GAIIx, HiSeq and MiSeq sequencing platform The NEXTflex DNA Sequencing Kit for Biomek FXp is designed to prepare single, paired-end and multiplexed Illumina -compatible genomic DNA libraries using the Biomek FXp Span8/MC hybrid liquid handler. Minimized setup time for high-throughput sequencing using the NEXTflex DNA Sequencing Kit for Biomek FXp means less labor time and more consistent sample preparation. The NEXTflex DNA Sequencing Kit features Enhanced Adapter Ligation Technology resulting in library preps with a larger number of unique sequencing reads. With Bioo Scientific s improved enzymatic ligation mix, ligations can be performed with longer adapters and better binding efficiencies. Output genomic DNA libraries are compatible with the Illumina GAIIx, HiSeq and MiSeq platforms. In addition, sets of up to 96 unique adapter barcodes are available for multiplexing. The library preparation is automated using the SPRIWorks HT protocol on the Beckman Coulter Biomek FXp Span8/MC hybrid liquid handler. DNA-Seq Library Prep - Illumina Compatible Competitive Analysis Table 4: Comparison of NEXTflex DNA-Seq Kit for Biomek and Competitor s Kits NEXTflex DNA Sequencing Kit for BioMek FXp Competitor X s Protocol Enhanced Adapter Ligation Technology Yes No Multiplex Barcodes 96 No Magnetic Bead Purification Yes Yes Compatible with SPRIWorks HT Protocol Yes Yes Gel-Free Protocol Yes Yes Price/Reaction Lower Higher NEXTflex DNA Sequencing Kit (BioMek FXp Compatible) 48 rxns NEXTflex DNA Barcodes rxns NEXTflex DNA Barcodes rxns NEXTflex DNA Barcodes rxns NEXTflex DNA Barcodes rxns NEXTflex-96 DNA Barcodes (in 96-well plate format) 768 rxns NEXTflex-96 DNA Barcodes (in tube format) 768 rxns 23

24 DNA-Seq Library Prep - Illumina Compatible NEXTflex DNA-Seq Modules for Illumina Flexible options The NEXTflex Modules are available in aliquots containing any number of reactions Enhanced Adapter Ligation Technology offers a larger number of unique sequencing reads Automation-friendly workflow is compatible with liquid handlers Convenient Reactions are provided in master mix format to reduce steps during DNA sample prep workflows Functionally validated with Illumina sequencing platforms Bioo Scientific now offers NEXTflex DNA, ChIP-Seq, and PCR-Free Modules allowing even more flexibility for NGS library prep. These modules are provided in a convenient master mix format to reduce steps during DNA sample prep workflows. Modules are available for each step in the library preparation protocol. Every NEXTflex Module passes rigorous enzymatic quality control and is functionally validated by sequencing on an Illumina platform. The NEXTflex Ligation modules incorporate Bioo Scientific s proprietary Enhanced Adapter Ligation Technology, which results in library preps with a larger number of unique sequencing reads. With Bioo Scientific s improved enzymatic ligation mix, ligations can be performed with longer adapters and better ligation efficiencies. NEXTflex DNA Modules 1 µg input requirement For use with genomic DNA samples These modules are also components of the NEXTflex DNA Sequencing Kit. The NEXTflex DNA Modules offer additional flexibility for next generation sequencing library preparation. They can be used to prepare single, paired-end and multiplexed genomic DNA libraries for sequencing. These modules should be used with the NEXTflex DNA Barcodes for multiplexing (see pg 26) NEXTflex DNA End Repair Mix 100 rxns NEXTflex DNA Adenylation Mix 100 rxns NEXTflex DNA Ligation Mix 100 rxns NEXTflex DNA PCR Master Mix 100 rxns NEXTflex ChIP-Seq Modules Low 10 ng input requirement For use with ChIP or genomic DNA samples End repair and ligation mixes are optimized for low DNA input The NEXTflex ChIP-Seq Modules offer additional flexibility for next generation sequencing library preparation. The NEXTflex ChIP-Seq Modules contain specially designed NanoQ enzymes and buffers needed for sample preparation of chromatin immunoprecipitated or regular genomic DNA for next generation sequencing NEXTflex ChIP Sequencing End Repair Mix 100 rxns NEXTflex ChIP Sequencing Adenylation Mix 100 rxns NEXTflex ChIP Sequencing Ligation Mix 100 rxns NEXTflex ChIP Sequencing PCR Master Mix 100 rxns 24

25 NEXTflex PCR-Free Modules µg input requirement For use with genomic DNA samples These modules are also components of the NEXTflex PCR-Free DNA Sequencing Kit. These modules should be used with the NEXTflex PCR- Free Barcodes for multiplexing (see pg 26) NEXTflex PCR-Free End Repair Mix 100 rxns NEXTflex PCR-Free Adenylation Mix 100 rxns NEXTflex PCR-Free Ligation Mix 100 rxns NEXTflex DNA-Seq Multiplexing Options for Illumina DNA-Seq Library Prep - Illumina Compatible The NEXTflex and NEXTflex-96 DNA Barcodes are adapters containing indexed sequences that offer an improved multiplexing workflow and flexible setup. This automation-friendly format enables multiplexing of up to 96 samples. The NEXTflex DNA Barcodes are available in sets of 6, 12, 24 and 48 unique adapters. For scientists who want to multiplex more than 48 samples, the NEXTflex-96 DNA Barcodes are sets of 96 barcodes that are available in microfuge tube or 96-well plate formats. The ability to pool samples in an efficient way significantly decreases hands-on time while providing robust data quality. Figure 8. NEXTflex Adapter Design Read 1 DNA Insert Index A Read 2 B The NEXTflex adapters contain the full complement of sequencing flow cell binding regions (A, B), which eliminates the need to perform PCR to add the barcode tag. Figure 9. Deep Sequencing of NEXTflex DNA Barcode Generated Sequencing Libraries. Libraries generated using the NEXTflex DNA Barcodes have a consistent percentage of usable reads

26 DNA-Seq Library Prep - Illumina Compatible The NEXTflex Barcodes simplify high-throughput sequencing with up to 96 available adapters. Using the NEXTflex DNA Sequencing Kit, 48 libraries were prepared and tagged with a different barcoded adapter. The percentage of index reads that could be used is represented by the height of the bars in Figure 5. Most barcoded adapters were read perfectly. The NEXTflex index utilizes double error correction, ensuring that single base changes during phasing do not unrecognizably alter the barcode sequence. Barcodes for NEXTflex ChIP-Seq & Rapid DNA-Seq Kits NEXTflex ChIP Sequencing Barcodes rxns NEXTflex ChIP Sequencing Barcodes rxns NEXTflex ChIP Sequencing Barcodes rxns NEXTflex ChIP Sequencing Barcodes rxns NEXTflex ChIP Sequencing Barcodes rxns Barcodes for NEXTflex Rapid DNA-Seq, DNA-Seq & Methyl-Seq 1 Kits NEXTflex DNA Barcodes rxns NEXTflex DNA Barcodes rxns NEXTflex DNA Barcodes rxns NEXTflex DNA Barcodes rxns NEXTflex-96 DNA Barcodes 768 rxns Barcodes for NEXTflex PCR-Free DNA-Seq Kits NEXTflex PCR-Free Barcodes rxns NEXTflex PCR-Free Barcodes rxns NEXTflex PCR-Free Barcodes rxns NEXTflex PCR-Free Barcodes rxns Barcodes for NEXTflex Bisulfite-Seq Kits NEXTflex Bisulfite Sequencing Barcodes rxns NEXTflex Bisulfite Sequencing Barcodes rxns 26

27 AIR DNA-Seq Multiplexing Options for Illumina AIR DNA Barcodes contain 12, 24, 36 or 48 unique barcodes, which can be used to provide flexibility in high-throughput sequencing applications. They significantly increase scale and throughput while reducing costs by allowing the user to pool up to 48 multiple library preparations into a single flow cell. The AIR DNA Barcodes accomplish this by using a 6 nt index on the PCR primer. This allows for proper differentiation between samples, preventing poor reads from single base errors introduced during PCR AIR DNA Barcodes rxns AIR DNA Barcodes rxns AIR DNA Barcodes rxns AIR DNA Barcodes rxns DNA-Seq Library Prep - Illumina Compatible Imagine how much money and time you could save with your NGS library preparation. KEY FEATURES Pre-programmed with NEXTflex library prep protocols Fully automated, hands-off NGS library prep Open platform Scalable for processing of up to 96 samples in parallel See page 54 for more information. 27

28 28

29 DNA-Seq Library Prep Ion PGM & Ion Proton Compatible 29

30 DNA-Seq Library Prep - Ion PGM & Ion Proton Compatible NEXTflex DNA Sequencing Kits for Ion PGM & Ion Proton Flexible barcode options - Kits contain 8, 16, 32 or 64 unique barcodes Simplified workflow with 96-well plate compatible volumes Compatible with DNA-Seq & ChIP-Seq Compatible with Life Technologies Ion Torrent sequencing platform The NEXTflex DNA Sequencing Kit for Ion PGM and Ion Proton is designed to prepare genomic DNA or ChIP-Seq libraries for sequencing using Life Technologies Ion PGM or Ion Proton sequencing platform. The NEXTflex DNA Sequencing Kit features Enhanced Adapter Ligation Technology, resulting in library preps with a larger number of unique sequencing reads. This NEXTflex Kit simplifies workflow by using master mixed reagents and bead-based cleanup. In addition, the ability to perform the reactions in a 96-well plate makes this kit more automationfriendly than other commercially available kits. Competitive Analysis Table 5: Comparison between NEXTflex and Competitor s DNA Library Prep Kits for the Ion PGM & Ion Proton NEXTflex DNA Sequencing Kit Competitor X s Protocol Enhanced Adapter Ligation Technology Yes No 96-Well Plate Compatible Yes No Automation Friendly Yes Less So Size Selection Components Included Yes No Starting Material Required 10 ng - 1 µg 10 ng - 1 µg NEXTflex DNA-Seq Multiplexing Options for Ion PGM & Ion Proton The NEXTflex DNA Barcodes for Ion PGM and Ion Proton are adapters containing indexed sequences that offer an improved multiplexing workflow and flexible setup. This format enables multiplexing of up to 64 samples. The NEXTflex DNA Barcodes are available in sets of 8, 16, 32 and 64 unique adapters. The ability to pool samples in an efficient way significantly decreases hands-on time while providing robust data quality NEXTflex DNA Sequencing Kit 8 rxns NEXTflex DNA Sequencing Kit 48 rxns NEXTflex DNA Barcodes rxns NEXTflex DNA Barcodes rxns NEXTflex DNA Barcodes rxns NEXTflex DNA Barcodes rxns 30

31 NEXTflex DNA Sequencing Kit Protocol P1 Adapter P1 Adapter GENOMIC DNA FRAGMENT END REPAIR ADD ADAPTERS LIGATION A Adapter CTGA GACT Key Sequence A Adapter CTGA GACT 30 Minutes (Optional stop point) 15 Minutes (Optional stop point) DNA-Seq Library Prep - Ion PGM & Ion Proton Compatible P1 Adapter SIZE-SELECTION A Adapter CTGA GACT 1-2 Hours (Optional stop point) P1 Adapter PCR GACT CTGA GACT A Adapter 1 Hour (Optional stop point) DILUTION FACTOR DETERMINATION BIOANALYZER AND/OR qpcr DNA TEMPLATE PREPARATION 31

32 32

33 DNA-Seq Library Prep SOLiD Compatible 33

34 DNA-Seq Library Prep - SOLiD Compatible NEXTflex DNA Sequencing Kits for 5500 SOLiD Simplified workflow with 96-well plate compatible volumes Flexible barcode options - Barcode kits contain 16 to 96 unique barcodes Simplified workflow with master mixes reduces hands-on time Compatible with ChIP-Seq The NEXTflex DNA Sequencing Kit for 5500 SOLiD is designed to prepare single, paired-end and multiplexed genomic DNA or ChIP-Seq libraries for sequencing using Life Technologies 5500 SOLiD sequencing platform. The NEXTflex DNA Sequencing Kit for 5500 SOLiD features Enhanced Adapter Ligation Technology, resulting in library preps with a larger number of unique sequencing reads. This NEXTflex kit simplifies workflow by using master mixed reagents and bead-based cleanup. In addition, the ability to perform the reactions in a 96-well plate makes this kit more automation-friendly than other commercially available kits. Competitive Analysis Table 6: Comparison between NEXTflex and Competitor s DNA Library Prep Kits NEXTflex DNA Sequencing Kit Competitor X s Protocol Enhanced Adapter Ligation Technology Yes No 96-Well Plate Compatible Yes No Automation Friendly Yes Less So Size Material Required 10 ng - 1 µg 10 ng - 1 µg NEXTflex DNA Barcodes for 5500 SOLiD The NEXTflex DNA Barcodes for 5500 SOLiD are adapters containing indexed sequences that offer an improved multiplexing workflow and flexible setup. This format enables multiplexing of up to 96 samples. The NEXTflex DNA Barcodes for 5500 SOLiD sets A - E contain 8 rxns each of 16 unique barcodes; set F contains 8 rxns each of all 96 barcodes NEXTflex DNA Sequencing Kit for 5500 SOLiD 12 rxns NEXTflex DNA Sequencing Kit for 5500 SOLiD 48 rxns NEXTflex DNA Barcodes Set A for 5500 SOLiD 128 rxns NEXTflex DNA Barcodes Set B for 5500 SOLiD 128 rxns NEXTflex DNA Barcodes Set C for 5500 SOLiD 128 rxns NEXTflex DNA Barcodes Set D for 5500 SOLiD 128 rxns NEXTflex DNA Barcodes Set E for 5500 SOLiD 128 rxns NEXTflex DNA Barcodes Set F for 5500 SOLiD 128 rxns NEXTflex DNA Barcodes Set G for 5500 SOLiD 768 rxns 34

35 NEXTflex DNA Sequencing Kit Protocol GENOMIC DNA FRAGMENT END REPAIR BEAD SIZE-SELECTION 30 Minutes 30 Minutes (Optional stop point) DNA-Seq Library Prep - SOLiD Compatible ADD A 30 Minutes P1 Adapter ADD ADAPTERS Standard Adapter P1 Adapter LIGATION Standard Adapter 30 Minutes (Optional stop point) NICK TRANSLATION & PCR PCR Primer 2 1 Hour (Optional stop point) PCR Primer 1 DNA QUANTIFICATION & SIZE DISTRIBUTION (qpcr & BIOANALYZER) EMULSION PCR 35

36 DNA-Seq Library Prep - SOLiD Compatible NEXTflex DNA Sequencing Kits for SOLiD 4 Simplified workflow with 96-well plate compatible volumes Flexible barcode options - Barcode kits contain 16 to 96 unique barcodes Simplified workflow with master mixes reduces hands-on time Compatible with ChIP-Seq The NEXTflex DNA Sequencing Kit for SOLiD 4 is designed to prepare single, paired-end and multiplexed genomic DNA or ChIP-Seq libraries for sequencing using Life Technologies SOLiD 4 sequencing platform. The NEXTflex DNA Sequencing Kit for SOLiD 4 features Enhanced Adapter Ligation Technology, resulting in library preps with a larger number of unique sequencing reads. This NEXTflex kit simplifies workflow by using master mixed reagents and gel-based cleanup. In addition, the ability to perform the reactions in a 96-well plate makes this kit more automation-friendly than other commercially available kits. Competitive Analysis Table 7: Comparison between NEXTflex and Competitor s DNA Library Prep Kits NEXTflex DNA Sequencing Kit Competitor X s Protocol Enhanced Adapter Ligation Technology Yes No 96-Well Plate Compatible Yes No Automation Friendly Yes Less So Size Material Required 10 ng - 1 µg 10 ng - 1 µg NEXTflex DNA Barcodes for SOLiD 4 The NEXTflex DNA Barcodes for SOLiD 4 are adapters containing indexed sequences that offer an improved multiplexing workflow and flexible setup. This format enables multiplexing of up to 96 samples. The NEXTflex DNA Barcodes for SOLiD 4 sets A - E contain 8 rxns each of 16 unique barcodes; set F contains 8 rxns each of all 96 barcodes NEXTflex DNA Sequencing Kit for SOLiD 4 12 rxns NEXTflex DNA Sequencing Kit for SOLiD 4 48 rxns NEXTflex DNA Barcodes Set A for SOLiD rxns NEXTflex DNA Barcodes Set B for SOLiD rxns NEXTflex DNA Barcodes Set C for SOLiD rxns NEXTflex DNA Barcodes Set D for SOLiD rxns NEXTflex DNA Barcodes Set E for SOLiD rxns NEXTflex DNA Barcodes Set F for SOLiD rxns NEXTflex DNA Barcodes Set G for SOLiD rxns 36

37 NEXTflex DNA Sequencing Kit Protocol GENOMIC DNA FRAGMENT END REPAIR BEAD SIZE-SELECTION 30 Minutes 30 Minutes (Optional stop point) DNA-Seq Library Prep - SOLiD Compatible P1 Adapter ADD ADAPTERS P2 Adapter P1 Adapter LIGATION P2 Adapter 30 Minutes (Optional stop point) NICK TRANSLATION & PCR PCR Primer 2 PCR Primer 1 DNA QUANTIFICATION & SIZE DISTRIBUTION (qpcr & BIOANALYZER) EMULSION PCR 37

38 38

39 RNA-Seq Library Prep Illumina Compatible 39

40 RNA-Seq Library Prep - Illumina Compatible NEXTflex qrna-seq Kit for Construction of Molecular Indexed Libraries Enables high precision measurement of RNA concentration Enhanced Adapter Ligation Technology offers a larger number of unique sequencing reads Input ng of mrna or rrna-depleted total RNA Embedded sample index barcodes for multiplexing of up to 96 samples Automation-friendly workflow is compatible with liquid handlers Functionally validated with Illumina sequencing platforms Bioo Scientific s patent pending NEXTflex qrna-seq Kit, enables high precision gene expression analysis by RNA-Seq. Developed in conjunction with Cellular Research Inc., this new kit efficiently generates libraries equivalent to conventional RNA-Seq libraries, but with the added feature of molecular indexing. Similar to conventional RNA-Seq, sample RNA is converted to cdna fragments. Prior to PCR amplification, all cdna fragment ends are ligated to pairs of adapters chosen at random from a total set of 9,216 molecular indices. Individual DNA molecules of identical sequence become distinct through indexing (Figure 10), allowing for differentiation between re-sampling of the same molecule and sampling of a different molecule of identical sequence. Analysis using molecular indexing information provides an absolute, digital measurement of gene expression levels, irrespective of common amplification distortions observed in many RNA-Seq experiments. Figure 10. An illustration with 8 mapped reads to an mrna transcript either with or without molecular indexing. Individual molecular indices at fragment ends are represented by different colors. The number of cdna fragments represented by the reads is unknown without molecular indexing, but can be determined with molecular indexing. NEXTflex qrna-seq With Molecular Indexing Conventional RNA-Seq Without Molecular Indexing 8 reads 4 fragments mrna 8 reads 6 fragments 8 reads Unknown # of fragments 8 reads 8 fragments Stochastic Labeling of Individual DNA Molecules The NEXTflex qrna-seq Kit contains two sets of 96 distinct molecular labels on the sequencing adapters. Each label or index consists of an 8 nucleotide barcode tag. In the ligation reaction, these 96 adapters are present in vast molar excess over the concentration of the cdna fragments, and therefore serve as a non-depleting reservoir of molecular labels. Each end of a cdna fragment independently and randomly chooses and ligates to a single label from this pool of 96 adapters to result in a total of 96 x 96 = 9,216 possible combinations across both ends. For every clone sequenced, paired-end reads reveal the chosen label on each end along with adjoining cdna sequence. In addition to encoding DNA fragments at the molecular level, the kit also allows for the application of sample-specific barcodes during the library preparation PCR step. A more detailed description of the use of molecular indexing for RNA-Seq is available in our product application note. Protocol Using the NEXTflex qrna-seq Kit, mrna or rrna-depleted RNA is fragmented using a cationic buffer. Fragmented RNA undergoes first and second strand synthesis, followed by end-repair, adenylation, ligation and PCR. 40

41 POLY A SELECTED RNA FRAGMENTATION FIRST STRAND cdna SYNTHESIS SECOND STRAND SYNTHESIS RNA-Seq Library Prep - Illumina Compatible END REPAIR ADENYLATION ADD MOLECULAR ENCODING ADAPTERS ADAPTER LIGATION AND LIBRARY ENRICHMENT PCR Read 2 Read 1 Sample 1 CLUSTER GENERATION AND SEQUENCING Multiplexing NEXTflex qrna-seq Kits include barcodes. The 8 rxn kit includes 4 barcodes for multiplexing. Four different versions of the 48 reaction kit are available, each containing 24 unique barcodes. Up to 96 samples can be multiplexed at once NEXTflex qrna-seq Kit rxns NEXTflex qrna-seq Kit Set A 48 rxns NEXTflex qrna-seq Kit Set B 48 rxns NEXTflex qrna-seq Kit Set C 48 rxns NEXTflex qrna-seq Kit Set D 48 rxns 41

42 For automated solutions see page 54. RNA-Seq Library Prep - Illumina Compatible NEXTflex RNA Sequencing Kits for Illumina Enhanced Adapter Ligation Technology offers a larger number of unique sequencing reads Input ng of mrna or rrna depleted total RNA Up to 96 barcodes available for multiplexing contain embedded index sequences Automation-friendly workflow is compatible with liquid handlers Functionally validated with GAIIx, HiSeq and MiSeq platforms The patent pending NEXTflex RNA-Seq Kits provide an easy and flexible solution for generating single, paired-end and multiplexed libraries from mrna or rrna depleted total RNA. Using the NEXTflex RNA-Seq Kits, mrna or rrna depleted RNA is fragmented using a cationic buffer. Fragmented RNA undergoes first and second strand synthesis, followed by end-repair, adenylation, ligation and PCR. In addition, the availability of up to 96 unique adapter barcodes makes this the most high-throughput kit available for RNA-Seq. Competitive Analysis Table 8: Comparison between NEXTflex and Competitor s RNA Library Prep Kits NEXTflex DNA Sequencing Kit Competitor X s Protocol Enhanced Adapter Ligation Technology Yes No Multiplex Barcodes Available Up to 96 unique 96 dual index Automation Friendly Yes Yes log S12_BioO_14_AGTTCC observed ERCC expected vs observed Figure 11. Relative Levels of ERCC Control RNAs in RNA-Seq Libraries. Made from NEXTflex Poly(A) Bead Selected and Total RNA Correlation between expected and observed values for percentage of reads mapping to 94 ERCC control RNAs in RNA-Seq library made using 20 µl of NEXTflex Poly(A) Beads log ERCC expected 42

43 Figure 12. Relative Levels of Different Categories of Endogenous Transcripts Detected in RNA-Seq Libraries made from NEXTflex Poly(A) Bead Selected and Total RNA. Percentage of reads mapping to rrna regions and regions of interest with and without Poly(A) bead selection. 100% 90% 80% 70% 60% unaligned intergenic intron exon misc_rna lincrna mirna Mito_gDNA_rRNA_tRNA rrna RNA-Seq Library Prep - Illumina Compatible 50% 40% 30% 20% 10% 0% Total RNA (A549) Total RNA (A549) Total RNA (293) Total RNA (293) Bioo Beads 20 µl Bioo Beads 50 µl Bioo Beads 100 µl NEXTflex RNA Sequencing Kit 8 rxns NEXTflex RNA Sequencing Kit 48 rxns NEXTflex RNA Sequencing Barcodes rxns NEXTflex RNA Sequencing Barcodes rxns NEXTflex RNA Sequencing Barcodes rxns NEXTflex RNA Sequencing Barcodes rxns NEXTflex-96 RNA Sequencing Barcodes (in 96-well plate format) 768 rxns 43

44 RNA-Seq Library Prep - Illumina Compatible NEXTflex RNA Sequencing Kit Protocol FRAGMENTATION POLY (A) OR rrna DEPLETED RNA FIRST STRAND SYNTHESIS SECOND STRAND SYNTHESIS 10 Minutes 75 Minutes 60 Minutes (Optional Stop Point) END-REPAIR 30 Minutes (Optional Stop Point) ADD A 30 Minutes ADD ADAPTERS LIGATION 15 Minutes (Optional Stop Point) PCR 1 Hour (Optional Stop Point) BEAD CLEANUP 44

45 NEXTflex Directional RNA-Seq Kits (dutp-based) v2 for Illumina Offers measurement of mrna strand orientation dutp-based stranded version of the NEXTflex RNA-Seq Kit Detection of antisense transcription Combined steps for less sample loss Allows for single read or paired end transcriptome sequencing Input ng of mrna or rrna depleted total RNA Up to 96 barcodes available for multiplexing contain embedded index sequence Automation-friendly workflow is compatible with liquid handlers Functionally validated with GAIIx, HiSeq and MiSeq platforms 2 hours faster than the Directional RNA-Seq Kit (dutp-based) The NEXTflex Directional RNA-Seq Kit (dutp-based) v2 provides an easy and flexible solution for generating single read, paired-end and multiplexed libraries from mrna or rrna depleted total RNA. The NEXTflex Directional RNA-Seq Kit (dutp-based) v2 can be used to obtain stranded information which identifies the specific DNA strands a given RNA transcript was derived from. This stranded information improves transcript annotation and improves read alignment, reducing per sample sequencing costs. dutp-based strand orientation also enables the detection of antisense expression regulatory relationships. This kit was designed to be used in conjunction with the NEXTflex RNA-Seq Barcodes or NEXTflex-96 RNA-Seq Barcodes for multiplexing. The availability of up to 96 unique adapter barcodes makes this the most high-throughput kit available for RNA-seq and also allows for both single-read and paired end sequencing. RNA-Seq Library Prep - Illumina Compatible Figure to 3 Sequence Coverage. Normalized coverage by position. For each library, the average coverage is shown at each relative position along the transcripts length. 70 Gene Express 5'~3' bias Mean Coverage Gene % Magnetic Beads for mrna Purification The NEXTflex Poly(A) Beads (pg 47) now provide a convenient method for batch purification of pure, intact mrna upstream of NEXTflex Directional RNA-Seq library preparation. NEXTflex Poly(A) Beads beads use oligo(dt) primer to isolate polyadenylated messenger RNAs from 1 µg 10 µg of previously isolated total RNA NEXTflex Directional RNA-Seq Kit (dutp-based) v2 8 rxns NEXTflex Directional RNA-Seq Kit (dutp-based) v2 48 rxns NEXTflex RNA Sequencing Barcodes rxns NEXTflex RNA Sequencing Barcodes rxns NEXTflex RNA Sequencing Barcodes rxns NEXTflex RNA Sequencing Barcodes rxns NEXTflex-96 RNA Sequencing Barcodes (in 96-well plate format) 768 rxns 45

46 RNA-Seq Library Prep - Illumina Compatible NEXTflex Directional RNA-Seq (dutp-based) v2 Protocol FRAGMENTATION POLY (A) OR rrna DEPLETED RNA FIRST STRAND SYNTHESIS SECOND STRAND SYNTHESIS 10 Minutes 75 Minutes 60 Minutes (Optional Stop Point) ADD A 30 Minutes ADD ADAPTERS LIGATION 15 Minutes (Optional Stop Point) UDG TREATMENT & PCR 1 Hour (Optional Stop Point) BEAD CLEANUP NEXTflex Directional RNA-Seq Kits (dutp-based) for Illumina The patent pending NEXTflex Directional RNA-Seq Kit (dutp-based) provides an easy and flexible solution for generating single, paired-end and multiplexed libraries from mrna or rrna depleted total RNA. This kit is compatible with the NEXTflex RNA-Seq Barcodes and the NEXTflex-96 RNA-Seq Barcodes (pg 51). This version will be phased out at the end of 2013, replaced with the NEXTflex Directional RNA-Seq Kit (dutp-based) v2 (pg 45) NEXTflex Directional RNA-Seq Kit (dutp-based) 8 rxns NEXTflex Directional RNA-Seq Kit (dutp-based) 48 rxns 46

47 NEXTflex RNA-Seq & Directional RNA-Seq Modules for Illumina Flexible options The NEXTflex Modules are available in aliquots containing any number of reactions you need Enhanced Adapter Ligation Technology offers a larger number of unique sequencing reads Automation-friendly workflow is compatible with liquid handlers Convenient Reactions are provided in master mix format to reduce steps during RNA sample prep workflows Functionally validated with Illumina sequencing platforms Bioo Scientific now offers NEXTflex RNA-Seq Modules, offering even more flexibility to NGS library prep. These modules are provided in a convenient master mix format to reduce steps during DNA sample prep workflows. Modules are available for each step in the library preparation protocol NEXTflex RNA Fragmentation Mix 100 rxns NEXTflex RNA-Seq First Strand Synthesis Mix 100 rxns NEXTflex RNA-Seq Second Strand Synthesis Mix 100 rxns NEXTflex RNA-Seq End Repair Mix 100 rxns NEXTflex RNA-Seq Adenylation Mix 100 rxns NEXTflex RNA-Seq Ligation Mix 100 rxns NEXTflex RNA-Seq PCR Mix 100 rxns NEXTflex Directional First Strand Synthesis Mix 100 rxns NEXTflex Directional Second Strand Synthesis Mix 100 rxns NEXTflex RNA-Seq UDG Mix 100 rxns RNA-Seq Library Prep - Illumina Compatible NEXTflex Poly(A) Beads µg total starting RNA Conserve resources by selecting for mrna prior to library prep Magnetic bead-based protocol for easier wash step and higher recovery No organic solvents or precipitation step required Validated for NGS library preparation The NEXTflex Poly(A) Beads provide a convenient method for batch purification of pure, intact mrna upstream of library preparation for RNA- Seq. NEXTflex Poly(A) Beads beads use oligo(dt) primer to isolate polyadenylated messenger RNAs from 0.1 µg 10 µg of previously isolated total RNA. Separation using a magnetic stand allows for high mrna recovery. The intact mrna is eluted in small volumes, eliminating the need for precipitation. Ease of use and consistent results make the NEXTflex Poly(A) Beads ideal for NGS library preparation applications. These beads have been validated with: NEXTflex RNA-Seq Kits & NEXTflex RNA-Seq Modules NEXTflex Directional RNA-Seq Kits & NEXTflex Directional RNA-Seq Modules NEXTflex qrna-seq Kits NEXTflex Poly(A) Beads 8 rxns NEXTflex Poly(A) Beads 48 rxns NEXTflex Poly(A) Beads 100 rxns 47

48 RNA-Seq Library Prep - Illumina Compatible NEXTflex Small RNA Sequencing Kits for Illumina Eliminate ligation associated bias Flexible barcode options with 48 barcodes available for multiplexing Utilizes AIR Ligase, a highly efficient truncated T4 RNA Ligase for greater sequencing depth Simplified workflow reduces hands-on time Automation-friendly workflow is compatible with liquid handlers Functionally validated with the Illumina GAIIx, HiSeq, and MiSeq sequencing platforms The NEXTflex Small RNA Sequencing Kits provide an easy, flexible, cost-effective solution for generating single and multiplexed libraries from small RNA using Illumina GAIIx, HiSeq, and MiSeq sequencing platforms. This kit contains a specialized 3 adenylated adapter that specifically ligates onto micrornas and other small RNAs containing a 3 hydroxyl group. After adapter ligation and reverse transcription, libraries are amplified using a barcoded primer, allowing up to 48 samples to be multiplexed and sequence variations interrogated. The NEXTflex Small RNA Sequencing Kit incorporates AIR Ligase, an enhanced, truncated T4 RNA Ligase, which increases the efficiency with which small RNAs are tagged with adapter, giving greater sequence depth. The NEXTflex Small RNA Sequencing Kit contains all the material to prepare 24 or 48 single-plex libraries. In combination with the NEXTflex Small RNA Barcodes (see page 51 for more information), 48 samples can be profiled and studied simultaneously. In addition to small RNA sequencing, the kit is compatible with RIP-Seq and CLIP-Seq protocols. Competitive Analysis Table 10: Comparison between NEXTflex and Competitor s Small RNA-Seq Library Prep Kits NEXTflex Small RNA-Seq Kit Competitor X s Protocol Length of Protocol Very Short Long AIR Ligase Yes No Automation Friendly Yes No All Enzymes Included Yes No Eliminate Ligation Associated Bias Figure 14. Correlation of mirna sequencing and microarray data using randomized adapter ligation technology (a) and using a conventional competitor s small RNA library preparation procedure (b). A Read Count Rank fnn-enn mir-20a 10 mir mir-18a mir-20a mir-16 mir-92a 7 mir-17 8 mir-103 mir-19b 7 6 mir-92a mir mir-17 mir-18a 5 mir-19b 4 mir-19a 4 mir-19a 3 3 let-7a 2 mir-106b 2 let-7a R 2 = mir-106b R 2 = Microarray Probe Signal Rank Microarray Probe Signal Rank B Read Count Rank fnn Bioo Scientific s patent pending, randomized adapters allow for bias-free ligation, allowing the user to determine true representation and expression profiles of small RNAs. 48

49 Figure 15. Mouse mirna Lung Expression Levels Normalized Expression as a % of Total MicroRNA microrna.org Database Results Bioo Scientific Results mmu-let7a mmu-mir-126 mmu-let7a mmu-let7b mmu-mir-26 mmu-mir-27 mmu-mir-21 mmu-mir-16 mmu-mir-143 mir126-5p Figure 16. Mouse mirna Heart & Muscle Expression Levels Heart Heart mmu-mir-143 mmu-mir-1-2 mmu-mir-1-1 mmu-mir-133b mmu-mir-133a-2 mmu-mir-133a-1 mmu-mir-451 mmu-mir-30 mmu-mir Muscle Muscle mmu-mir-101a mmu-mir-143 mmu-mir-30e mmu-mir-133b mmu-mir-30a mmu-mir-1-1 mmu-mir-378 RNA-Seq Library Prep - Illumina Compatible Citations: Jayaprakash, A. D., Jabado O., Brown, B. D. and Sachidanandam, R. (Sept 2, 2011), Identification and remediation of biases in the activity of RNA ligases in small-rna deep sequencing Nuc Acid Res, doi: /nar/gkr693 Nakashe, P. et al. (2011) Adapter Dimer Reduction in High-Throughput microrna Profiling. OMICS 1(1): NEXTflex Small RNA Sequencing Kit 24 rxns NEXTflex Small RNA Sequencing Kit 48 rxns NEXTflex Small RNA Barcodes (Set A) 96 rxns NEXTflex Small RNA Barcodes (Set B) 96 rxns NEXTflex Small RNA Barcodes (Set C) 96 rxns NEXTflex Small RNA Barcodes (Set D) 96 rxns 49

50 RNA-Seq Library Prep - Illumina Compatible NEXTflex Small RNA Sequencing Protocol 5 Adapter RNA NNNN 3 LIGATION NNNN 5 LIGATION (P) 3 Adenylated Adapter NNNN 3 Adenylated Adapter Ligated RNA REVERSE TRANSCRIPTION 1st Strand Synthesis Product RT primer NNNN NNNN 5 and 3 Adapter Ligated RNA FIRST PCR & SIZE SELECTION 1st Strand Synthesis Product Universal Primer Barcode Primer 1 PCR Product SECOND PCR & SIZE SELECTION Size-selected PCR Product Primer 1 Primer 2 Final PCR Product 50

51 RNA-Seq Multiplexing Options for Illumina The NEXTflex RNA-Seq Barcodes and NEXTflex-96 RNA-Seq Barcodes are automation-friendly adapters that are designed for use with the NEXTflex RNA-Seq Kit (see page 42) and the NEXTflex Directional RNA-Seq Kit (dutp-based) v2 (see page 45). These adapters allow multiplexing of up to 96 unique samples in a single run for higher throughput. The ability to pool samples significantly decreases hands on time while preserving robust data quality. The NEXTflex RNA-Seq Barcodes are available in sets of 6, 12, 24 and 48 unique adapters. The NEXTflex-96 RNA-Seq Barcodes are sets of 96 barcodes that are available in microfuge tube or 96-well plate formats NEXTflex RNA-Seq Barcodes rxns NEXTflex RNA-Seq Barcodes rxns NEXTflex RNA-Seq Barcodes rxns NEXTflex RNA-Seq Barcodes rxns NEXTflex-96 RNA-Seq Barcodes 768 rxns RNA-Seq Library Prep - Illumina Compatible Barcodes for NEXTflex Small RNA-Seq Kits Four sets of 12 NEXTflex Small RNA Barcodes with embedded index sequences are available, offering an improved multiplexing workflow and increased flexibility. These barcodes are designed for use with the NEXTflex Small RNA Sequencing Kit (see page 48). The ability to pool samples in an efficient way significantly decreases hands-on time while providing robust data quality. The primer barcoding system utilizes a 6 nt index to differentiate up to 48 different samples on a single flow cell lane. 48 barcoded primers are divided into 4 different kits (A-D) NEXTflex Small RNA Barcodes (Set A) 96 rxns NEXTflex Small RNA Barcodes (Set B) 96 rxns NEXTflex Small RNA Barcodes (Set C) 96 rxns NEXTflex Small RNA Barcodes (Set D) 96 rxns 51

52 52

53 Next-Gen Sequencing Automation 53

54 Equipment SEQBOT Liquid Handler Bioo Scientific s SEQBOT is a new liquid handling platform featuring an 8-channel pipette head, designed to perform up to 96 reactions per run. Fully supported recipes for Bioo Scientific kits are available on this platform, allowing the automation of these kits with no programming required. A variety of configurations are available to meet your specific needs and budget. The base kit provides an 8-channel liquid handler with pipette mixing. This kit includes a full personal computer running Windows 7 64-bit OS, contains all programs necessary for running Bioo Scientificprovided recipes, and allows development of custom programs. For increased throughput, several additional accessories are available, including a shaker nest for full-plate shaking, an automated magnetic stand to eliminate manual transfer of the plate on and off the magnet, and a plate crane for full walk-away automation. SEQBOT Accessory Guide Item Benefits Compatibility SEQBOT Basic liquid handler All NEXTflex Kits Plexiglas Enclosure Protects samples from environmental contamination All NEXTflex Kits Plate Shaker Replaces pipette mixing with 96-well simultaneous mixing, increases speed All NEXTflex Kits Automated Magnetic Stand Heated Plate Shaker Heated/Cooled Plate Shaker Crane Reduces labor, eliminates manual movement of library plate on/off magnetic stand Replaces pipette mixing with 96-well simultaneous mixing, increases speed, performs elevated temperature incubations Replaces pipette mixing with 96-well simultaneous mixing, increases speed, performs elevated temperature incubations, performs reduced temperature incubations Full walk-away automation possible, reduces labor by automatically replacing tip boxes and moving plates All NEXTflex Kits NEXTflex Rapid DNA-Seq Kit NEXTflex Rapid DNA-Seq Kit NEXTflex RNA-Seq Kit NEXTflex Directional RNA-Seq Kit NEXTflex qrna-seq Kit All NEXTflex Kits For purchasing information please call or at sales@biooscientific.com 54

55 Imagine how much money and time you could save with your NGS library preparation. Equipment KEY FEATURES Pre-programmed with NEXTflex library prep protocols Fully automated, hands-off NGS library prep Open platform Scalable for processing of up to 96 samples in parallel Automation for Other Liquid Handlers Recipe files are also available for automation of Bioo Scientific s most popular kits using additional liquid handler platforms, including the Beckman Coulter Biomek FX/FXp, Eppendorf epmotion, and Tecan evofreedom. Recipe files can be customized for your preferred deck layout and available accessories. If a recipe has not yet been developed for your kit on your platform, Bioo Scientific will work with you to develop a suitable solution. Simply us at nextgen@biooscientific.com for your liquid handler needs. 55

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57 DNA Fragmentation & Nucleic Acid Isolation for Next-Gen Sequencing 57

58 DNA Fragmentation & Nucleic Acid Isolation for NGS AIR DNA Fragmentation Kit Optimized to physically shear genomic DNA into uniform fragments of genomic DNA Offers 30% greater DNA recovery than competitor s fragmentation protocol Includes complete protocol and proprietary fragmentation buffer The AIR DNA Fragmentation Kit offers a cost-effective procedure for the fragmentation of genomic DNA for next-generation sequencing sample prep. It has been optimized to physically shear genomic DNA to produce uniform fragments. The AIR DNA Fragmentation Kit shears and recovers DNA more efficiently than traditional nebulization protocols allowing for smaller sample sizes. This kit contains nebulizers, a proprietary fragmentation buffer and complete instructions. It is designed to be used with DNA sample prep protocols for Illumina, Ion Torrent, SOLiD and Roche 454 sequencing. The procedure requires a compressed air source with a pressure regulator. Please see manual for complete details. Figure 17. Coverage Analysis of DNA Sheared using the AIR DNA Fragmentation Kit. Using the AIR DNA Fragmentation kit, 1µg of Pseudomonas aeruginosa genomic DNA was fragmented, paired end libraries were constructed, and sequence data generated using one lane of an Illumina GAIIx (2x51). There was an average of 159 reads per position and 92% of the reads mapped perfectly to Pseudomonas aeruginosa. Coverage 250 Reads per Position Reference Sequence Location AIR DNA Fragmentation Kit 10 rxns AIR DNA Fragmentation Kit 40 rxns 58

59 NextPrep Blood DNA Isolation Kit Designed for NGS applications Only six pipetting steps required Fast and easy DNA isolation from whole blood or buffy coat/wbcs No protease digestion step No organic extractions Validated with NEXTflex Sequencing Kits The NextPrep -Blood DNA Isolation Kit is designed for the rapid purification of genomic DNA from whole blood or buffy coat for use in next generation sequencing library preparation. Figure 18. Rapid 15 Minute DNA Isolation Protocol Lyse Centrifuge Wash x 2 Dry Elute DNA Fragmentation & Nucleic Acid Isolation for NGS DNA is recovered by solid phase extraction onto a silica matrix in a spin basket format, using a novel rapid protocol that results in extremely clean DNA. The entire procedure can be completed in ~ 15 minutes. Yields are usually several micrograms (2.5 µg 5 µg) of high molecular weight DNA with Abs 260/280 values between from a starting volume of 0.3 ml of whole blood. Yields can be increased to ~10 25 µg by using the modified protocol for buffy coat. The DNA is ideal for use as input for creating libraries for next generation sequencing. For this application we recommend Bioo Scientific s NEXTflex DNA-Seq Kits. The recommended anticoagulant is EDTA. Figure 19. DNA-Seq Library From Made Human Blood Using the NEXTprep -Blood DNA Isolation Kit [bp] DNA isolated from HemaCare Donor 5C NEXTflex DNA Sequencing Library DNA was fragmented by nebulization using AIR Fragmentation Kit. Fragmented DNA size-selected for bp fragments on 1.2% TAE agarose gel. Input DNA amount: ng Recovered: 30 µl of PCR product with a concentration of 1.08 ng/µl; ng total NEXTprep Blood DNA Isolation Kit 8 rxns NEXTprep Blood DNA Isolation Kit 48 rxns 59

60 DNA Fragmentation & Nucleic Acid Isolation for NGS BiooPure RNA Isolation Reagent Offers high yields of total RNA from tissues and cells Includes protocol for enrichment of microrna Works well for recovering RNA from cell-free fluids Includes inert co-precipitant for maximizing RNA recovery Liquid nitrogen not required More cost-effective than similar reagents The BiooPure RNA Isolation Reagent is a single-phase reagent for extraction of total RNA or enriched small RNA (including mirna) from solid tissues and cultured cells for use in next generation sequencing library preparation. The BiooPure reagent contains guanidinium, a powerful chaotropic agent effective for rapidly inactivating nucleases, and phenol, an organic solvent used to denature and separate proteins and DNA from RNA. Figure 20. Bioanalyzer trace of total RNA isolation using BiooPure [nt] Ladder Sample The bioanalyzer trace shows data using the RNA 6000 Pico Chip Total RNA Assay. The sample was purified from A549 human lung cancer cell line. The sample was at a concentration of 4-5 ng/µl before dilution to fall in the quantitative range of the chip. The RIN number for this sample is L BiooPure RNA Isolation Reagent 30 ml BiooPure RNA Isolation Reagent 90 ml 60

61 NEXTprep Small RNA Isolation Kit Ideal for enrichment of micrornas and other small RNAs from eukaryotic cells and tissues Uses spin column format Fast and easy small RNA extraction Offers high yields of small RNA Validated with NEXTflex Sequencing Kits The NEXTprep Small RNA Isolation Kit is designed for enrichment of small RNA from eukaryotic cells and tissues. The small RNA recovered has been verified as suitable for making small RNA libraries for next generation sequencing (NGS) using the NEXTflex Small RNA Sequencing Kit. The NEXTprep Small RNA Isolation Kit includes BiooPure RNA Extraction Reagent for initial disruption of tissues or cells. BiooPure is a singlephase reagent that contains guanidinium thiocyanate, a powerful chaotropic agent effective for rapidly inactivating nucleases, and phenol, an organic solvent used to denature and separate proteins and DNA from RNA. The kit also includes silica filter spin basket assemblies and tubes for final purification of small RNA by solid phase extraction. The kit allows recovery of small RNAs approximately 100 bases and smaller, with less than ~5% by mass of contamination with larger RNAs. Solid tissues or cells are disrupted by grinding or vortexing in BiooPure RNA Extraction Reagent. The resulting lysate is mixed with 1-bromo- 3-chloropropane and centrifuged, which results in phase separation with the RNA partitioned in the upper aqueous phase. The aqueous phase is recovered and mixed with ethanol suitable for selective precipitation of large RNA, which is removed by solid phase extraction on a silica filter. The flow-through containing small RNA is recovered and mixed with additional ethanol to precipitate the small RNA, which is then purified by solid phase extraction onto a second silica filter. The filter is washed and RNAs <~100 bases are eluted in RNase-free water containing a trace amount of EDTA. DNA Fragmentation & Nucleic Acid Isolation for NGS NEXTprep Small RNA Isolation Kit 10 rxns MiraZol Small RNA Isolation Kit High yields of small RNA <~ 100 bases Complete kit with all reagents except isopropanol Linear polyacrylamide co-precipitant included The MiraZol Small RNA Isolation Kit is similar to the NextPrep Small RNA Isolation Kit, but uses isopropanol precipitation in place of silica filter spin columns for purification of small RNA from cells and tissue NEXTprep Small RNA Isolation Kit 10 rxns 61