"Expanded applications for NGS library prep exploring rapid workflows with Nextera and challenging RNA samples" Todd Deppe Sample Prep Specialist Regional Marketing UCR Feb 11, 2014 2013 Illumina, Inc. All rights reserved. Illumina, IlluminaDx, BaseSpace, BeadArray, BeadXpress, cbot, CSPro, DASL, DesignStudio, Eco, GAIIx, Genetic Energy, Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, Infinium, iselect, MiSeq, Nextera, NuPCR, SeqMonitor, Solexa, TruSeq, TruSight, VeraCode, the pumpkin orange color, and the Genetic Energy streaming bases design are trademarks or registered trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.
What could you accomplish if you could sequence any bacterial genome in a single day for <$100? you could detect mutations below 5% frequency? you only needed 30 min hands on time to go from DNA to data? you only needed a single nanogram of genomic DNA for sequencing? 2"
Nextera XT DNA Sample Prep The fastest & easiest prep for small genomes, PCR amplicons and plasmids Rapid"Prep" - 90"min"prep,"only"15"min"of"hands"on"9me" Op9mized"for"small"genomes,"PCR"amplicons" and"plasmids" Innova9ve"sample"normaliza9on" - No"library"quan9fica9on"needed" Fastest"9me"to"results" - DNA"to"analyzed"data"in"<8"hours"with"MiSeq" Ultra"low"input" - only"a"single"nanogram"of"input"dna"needed" 3"
4" Step 1: Tagmentation of template DNA
5" Step 2: PCR to add adapters and indices
6" Step 3: Cleanup and Sequence
Sample Normalization is included No library quantification or qpcr is required go straight to MiSeq! Completed libraries, range of yields Index CV for 20-sample pools Pool A Pool B Quant in triplicate with qpcr Calculate dilutions Manually dilute and pool 15.8% 18.2% 5 µl of each desired library Bead-based Normalization: Bind, Wash, Elute 13.5% 15.5% Nextera XT sample pooling is as simple as pipetting 5 µl! 7"
Nextera XT Sample Prep Workflow Complete DNA to data in only 8 hr with MiSeq 1 ng input! No post-tag cleanup! Sample normalization included! New amplicon workflow! 8"
Amplicon Workflow for Nextera XT Sample Prep Run Set-up Sequence Data Analysis Feature New Nextera XT kit Benefits Fastest sample prep Fewest hands-on steps and minimal hands on time Feature New Nextera XT supported workflow Automated manifest file generation Benefits Minimal informatics skills to setup analysis Reduce errors in run setup Feature All-in-one sequence and analysis instrument Wizard driven UI Directed connected to BaseSpace Benefits No additional amplification/ computer hardware needed Easy to use Automated bioinformatics Feature New Nextera XT PCR amplicon bioinformatics analysis workflow Using BWA/GATK Benefits Automated data analysis with min bioinformatics skills Easy to read, biologically relevant reports Industry standard algorithms 9"
CE for Amplicons? Time to try NGS! Nextera XT enables easy transition to MiSeq FAST% - Prep"a"sample"in"<90"min"with"15" min"hands"on"9me" - Rapid"way"to"add"sequencing" adapters"for"ilmn"sequencers" EASY% - Prep"exis9ng"amplicons,"including" long"range">1"kb!" - Simple"sample"normaliza9on"for"easy" pooling" - Barcoding"supports"pooling"up"to"96" samples"per"lane" ENABLING%PRICE% - Single"Nextera"XT"prep"@"~$30"for"a" pool"of"many"amplicons" 10"
Nextera for PCR Amplicons Quick workflow for long or short range PCR PCR AMP Pool Amplicons, Nextera XT Pool Samples Miseq Run Short Range - CFTR 7 Exons 255-416 bp range 90 min total 15 min hands on 12 patient samples 2x36 (6.5hrs) 95%>Q30 Long Range BRCA1/2 22 Amplicons 1-6kb range 68.5kb total 90 min total 15 min hands on 5 patient samples 2x76(15hrs) 95%>Q30 11"
Nextera for PCR Amplicons Detection of mutations in CFTR with short range PCR Amplicons Het W1282X Het deletion Δ F508 Exon 10 Exon 20 Complete coverage of target regions at >300X depth Complete variant concordance Collaboration with Steven Abbs and Michale Yau, London 12"
Nextera for PCR Amplicons Detection of mutations in BRCA1 & 2 with long range PCR Complete coverage of target regions at >50x depth Complete variant concordance 13" Collaboration with Graham Taylor, Leeds, UK
Sequencing HIV Isolates with MiSeq and Nextera XT MiSeq changed the scales. With Sanger sequencing I could pick up a 50/50 mutation maybe 25/75, but I could never pick up a minor population around 2% like I can with MiSeq. There s a vast improvement in speed. Using the Nextera XT kit, our process went from several days to only half a day that s just 4 hours for 96 samples. We can monitor mutations pretty rapidly with the Nextera XT kit and MiSeq. 14"
Nextera XT is perfect for microbial genomes No bug is safe from MiSeq! FAST% - Prep"a"sample"in"<90"min"with"15"min" hands"on"9me" - Sequence"genomes"in"a"single"day"with" MiSeq" EASY% - Simple"prep"makes"it"easy"to"prepare" many"samples"at"the"same"9me" - Simple"sample"normaliza9on"for"easy" pooling" - Barcoding"supports"pooling"up"to"96" samples"per"lane" Please note, bacterial cells typically do not have teeth this white ENABLING%PRICE% - Sequence"a"bacterial"genome"for"<$100% with"xt"and"miseq!" 15"
Superior de novo bacterial sequencing with MiSeq Even coverage across challenging high GC genomes PGM exhibits poor coverage uniformity Nextera and MiSeq provide even coverage across entire genome Rhodobacter sphaeroides 4.6 Mb genome, 70% GC 16" Data courtesy of Dr. Tim Stinear, University of Melbourne, and Dr. Torsten Seemann, Monash University
Which version of Nextera should I use? Nextera Nextera XT Applications Large/complex genomes (human, mammalian, plants, inverts) Small genomes, amplicons, plasmids (bacteria, archea, viruses, PCR amplicons, plasmids) Total Sample Prep Time for 8 samples (hands on) 90 (15) 85 (10) DNA input 50 ng 1 ng Post tag cleanup? Zymo None Sample normalization Manual (e.g. bioanalyzer, qpcr) Bead-based normalization included Indexing 96 96 List Price per sample ~$75 ~$30 17"
Epicentre Solutions for challenging RNA seq Todd Deppe Sample Prep Specialist Regional Marketing 2013 Illumina, Inc. All rights reserved. Illumina, IlluminaDx, BaseSpace, BeadArray, BeadXpress, cbot, CSPro, DASL, DesignStudio, Eco, GAIIx, Genetic Energy, Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, Infinium, iselect, MiSeq, Nextera, NuPCR, SeqMonitor, Solexa, TruSeq, TruSight, VeraCode, the pumpkin orange color, and the Genetic Energy streaming bases design are trademarks or registered trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.
Key Product Groups DNA and RNA Sequencing PCR and RT-PCR Gene Expression Analysis In Vitro Transcription and RNA Research DNA and RNA Purification Cloning and Library Construction Transposomics Enzymes for Molecular Biology 19"
Enzyme Expertise Experience Over 25 years of production experience Unmatched technical and support expertise State of the art facilities for large-scale fermentation and purification Superior Enzymes Highly purified Tag-free enzymes ensuring native activity Thorough contaminant testing ensuring highest level of functional purity o DNA exonuclease & endonuclease o RNase o And others where necessary Excellent lot-to-lot consistency Very Flexible Options Many OEM opportunities with competitive pricing and rapid turnaround times Custom formulations including enzyme concentration, salts, detergents and reducing agents Glycerol-free options may also be available Multiple custom contaminant assays to choose from: o E. coli genomic DNA o Thiols o Non-specific proteases
21" Solutions for Life Science
The Illumina Value Chain: Total Workflow & Applications Solutions MasterPure" QuickExtract" TruSeq"&"ScriptSeq" TotalScript,"ART`Seq" Nextera" EpiGnome"(WGBS)" BaseSpace/" NextBio" Purifica:on% (rrna% Deple:on)% Library% Genera:on% Sequencing% Data% Analysis% Ribo`Zero" Globin`Zero" TS+Globin/RZ/PolyA" MiSeq,"HiSeq" Systems" 22"
Purification Molecular Biology Begins with Purification
Purification Success Begins With Purity Every Molecular Biology Application NGS Arrays Cloning 24"
Purification First Step: Purification TruSeq Custom Amplicon Nextera Nextera XT MasterPure Success Begins With Purity EpiGnome Ribo-Zero ScriptSeq Complete TruSeq Stranded RNA TruSeq mrna 25"
MasterPure Purifies Your Sample sputum thymus plasma kidney semen Buffy coat brain saliva heart urine liver blood 26"
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Purification MasterPure Advantages Sample MasterPure TM RNA Library Prep High Yields No Toxic Chemicals Retention of small RNA s Published with NGS, qpcr, and other molecular biology application. 28"
Purification MasterPure: Obtain Higher Yields Higher yields give you more sample options smaller samples are now possible One kit does it all no need to stock multiple kits Highest of any purification option 29"
Purification MasterPure: Don t Get Retracted MasterPure salting-out method retains sample complexity Other technologies eliminate small RNA s, mirna s 30"
Purification PCR Application: PCR Challenges and Solutions
Purification PCR FailSafe: Higher Yield and Specificity No more failed PCR results Traditional PCR Protocol: Isolate DNA Design Primers Every Sample Works Every Time Set Up Reaction Perform PCR Assess Results Trouble Spots Solved With FailSafe 32"
Purification PCR Solutions For All PCR Needs 33"
Purification PCR Don t take our word for it Site-directed mutagenesis Sub-cloning Gene detection Genotyping And many more Over 140 FailSafe citations "Epicentre products are great, as for FailSafe PCR, we use it all the time. Prof. Sir Martin Evans Nobel Laureate, Cardiff University Wales, UK 34"
Purification rrna Depletion Library Generation Sequencing Application: RNA-Seq More Informative Results
Purification rrna Depletion Library Generation Sequencing Limited Samples, of Variable Quality Many sample types are not abundant Many RNA samples are degraded RIN = 6 RIN = 5 RIN = 4 RIN = 3 Some RNA samples are neither abundant or high quality That s OK 36"
Purification rrna Depletion Library Generation Sequencing rrna Removal Produces More Informative Results ScriptSeq Complete Poly A More information retained With ScriptSeq Complete rrna Removal with ScriptSeq Complete - Keep ALL non-rrna s = more information in sample - Suitable for degraded or intact samples Poly A Selection Keep only mrna s with strongest AAA tails = less information in sample Lose all messages that aren t perfectly polyadenylated, spliced, capped, etc. 37"
Purification rrna Depletion Library Generation Sequencing Get Answers Faster ScriptSeq Complete Sample to cbot in < 1 day rrna Depletion Library Generation PCR (lunch) BioAnalyzer and Qubit Finished! 38"
Purification rrna Depletion Library Generation Sequencing Applications Cancer, Human Health [Human/Mouse/Rat Gold] New in 2013 Microbiology [Bacteria] Disease Studies; Tissue, Cells [Epidemiology] 39" Disease Studies; Blood [Globin-Zero] Yeast [Yeast] Agrigenomics [Plant Leaf, Seed/Root]
Purification rrna Depletion Library Generation Sequencing Flexible Kit Formats Available by itself as Ribo-Zero 40"
Purification rrna Depletion Library Generation Sequencing ScriptSeq Complete (Blood) Retain all genes New in 2013 Deplete rrna + globin mrna at same time Solution Available Only from Illumina/ Epicentre 41"
Purification rrna Depletion Library Generation Sequencing ScriptSeq Complete (Epidemiology) Retain host/pathogen transcriptomes Disease research studies Deplete human & bacterial rrna at same time Solution Available Only from Illumina/ Epicentre RNA-Seq is a tool for discovery Samples: Tissue, Cells, Biopsy, FFPE 42"
Purification rrna Depletion Library Generation Sequencing ScriptSeq Complete (Bacteria & Epidemiology) Deplete rrna from all types of bacteria even when you don t know their names 43"
Purification rrna Depletion Library Generation Sequencing Yeast Finally Get Their Own RNA-Seq Tools New in 2013! No Wasted Reads, Saves $$ 44"
Application: Low Input RNA-Seq Precious Samples Now Yield Answers
Purification rrna Depletion Library Generation Sequencing TotalScript: Rapid Workflow 9:00 10:00 11:00 noon 1:00 2:00 3:00 4:00 5:00 Only 1-5ng of Total RNA needed! Library Generation PCR (lunch) BioAnalyzer and Qubit Compatible with all Illumina Sequencing Platforms 46"
Purification rrna Depletion Library Generation Sequencing TotalScript: Choose Your Desired Results 2.6" 2.4" Approach Random Priming Mixed Priming Total RNA Input (ng) % rrna Coverage 1-5 <40% Even 1-5 <25% Slight 3 Bias dt Priming 1-5 <5% 3 Bias Normalized"Coverage" Normalized"Coverage" 2.6" 2.4" 2.2" 2" 1.8" 1.6" 1.4" 1.2" 1" 0.8" 2.2" 2" 1.8" 1.6" 1.4" 1.2" 1" 0.8" 0.6" 0.4" 0.2" 0" Random"Priming" 1" 5" 9" 13" 17" 21" 25" 29" 33" 37" 41" 45" 49" 53" 57" 61" 65" 69" 73" 77" 81" 85" 89" 93" 97" Length"of"RNA"5'"to"3'" Mixed"Priming" Choose your desired rrna content and transcript coverage 0.6" 0.4" 0.2" 0" 1" 4" 7" 10" 13" 16" 19" 22" 25" 28" 31" 34" 37" 40" 43" 46" 49" 52" 55" 58" 61" 64" 67" 70" 73" 76" 79" 82" 85" 88" 91" 94" 97" 100" Length"of"RNA"5'"to"3'" dt"priming" 2.6" 2.4" 2.2" Ribosomal Suppression Normalized"Coverage" 2" 1.8" 1.6" 1.4" 1.2" 1" 0.8" 0.6" 0.4" 0.2" 0" 1" 5" 9" 13" 17" 21" 25" 29" 33" 37" 41" 45" 49" 53" 57" 61" 65" 69" 73" 77" 81" 85" 89" 93" 97" Length"of"RNA"5'"to"3'" 47"
Purification rrna Depletion Library Generation Sequencing TotalScript: Species Tested 48"
Application: Ribosome Profiling Measure Gene Expression, Translational Control
ARTseq: Evolution of Next Generation Sequencing Active mrna Translation Sequencing 50"
What is Ribosome Profiling? In simplest terms next generation sequencing of ribosome protected mrna Also referred to as ribosome footprinting in literature Protein synthesis Ribosome translating mrna mrna Ribosome profiling: isolate and sequence only the mrna protected/actively translated by the ribosome 51"
Why is Ribosome Profiling Important? The ARTseq Kit produces RNA-Seq libraries from ribosome-protected mrna to: Investigate translational control. Measure gene expression. Identify translation start sites. Predict protein abundance. Investigate translational and co-translational processes in vivo. Ribosome profiling is a novel technique for investigating translational control. The method, based on deep sequencing of ribosome protected mrna fragments, provides a "snapshot" of the ribosomes active in a cell at a specific time point. This information can in turn be used to determine what proteins are being actively translated in a cell. Benefits Incorporates a rapid and simplified size-exclusion spin-column method to isolate monsomes. Compatible with yeast and mammalian samples. ARTseq libraries are compatible with Illumina Cluster Kits for single read, pairedend and multiplex sequencing using the Illumina GAII, HiSeq and MiSeq sequencers. Index-compatible: 12 Index Primers included in the kit. 52"
Why is Ribosome Profiling Important? Remember the Central Dogma of Molecular Biology: Ribosome profiling can be used as a proxy for protein expression - DNA to RNA to Protein RNA-seq gives information ONLY about mrna abundance Protein levels are a function of: - RNA stability - Regulation of translation - Protein stability 53"
Why is Ribosome Profiling Important? Powerful technique for investigating translational control Yields better estimate of protein abundance/expression than traditional RNA-seq Ribosome Profiling mrna-seq Correlation - Correlation - 54"
ARTseq: Optimized Sample Prep ARTseq = Active RNA Translation Lyse cells One Kit Solution! No Need to Optimize Protocol or MasterMix Reagents from Multiple Vendors! Sequence 55"
Epicentre Solution ARTseq = Active RNA Translation Optimized kits for mammalian and yeast samples Prepare Lysate Nuclease Digestion Size Exclusion Chromat. Ribo-Zero Isolate RPFs (PAGE) Library Prep ARTseq Ribosome Profiling Kits Only 2 PAGE purifications SEC instead of Sucrose gradients Master mixed reagents Optimized protocol Reproducible results 56"
ARTseq: Visualize Start, Stop Codons ARTseq CWP2 ORF Stop 3 UTR Results are enriched for ORFs in footprinted samples mrna-seq mrna-seq ARTseq Enriched for Protein Coding Sequences! 57"
Informatics guide also available from epibio.com There are 4 main steps in the ARTseq analysis pipeline: 1. Generation of fastq files 2. Trimming of adapters 3. Alignment to remove contaminants (optional) 4. Alignment to a reference genome and splice junctions using TopHat Following alignment, the resulting BAM file can be used for downstream analyses with other bioinformatics tools. In this document, we will demonstrate the use of the Cufflinks package1 for transcript annotation, assembly, quantification and visualization. 58"
Purification WGBS Sequencing Analysis Application: Epigenetics Whole Genome Bisulfite Sequencing
Purification WGBS Sequencing Analysis EpiGnome Methyl-Seq Whole Genome Bisulfite Sequencing now in reach for limited samples 60"
Purification WGBS Sequencing Analysis What is Epigenetics? Why are twins different? Twins with identical DNA are unique due to different epigenetic marks Over The Identical Twins time, epigenetic epigenetic begin twins life marks marks become with are turn nearly added different genes from.. on over identical and time off DNA choice the exercise environment stress of food A C T G C G A T C C G T A C G... A C T G C G A T C C G T A C G... 61"
Purification WGBS Sequencing Analysis DNA Methylation is Heritable Poor nutrition can impact three generations of chickens Frésard et al. Genetics Selection Evolution 2013 45:16 doi:10.1186/1297-9686-45-16 62"
Purification WGBS Sequencing Analysis Who Studies DNA Methylation? Growing area of research - Methyl-seq expected to surpass DNA-Seq by 2018 Stem cell research Animal genetics Cancer research Plant research 63"
Purification WGBS Sequencing Analysis Bisulfite Sequencing Identify Methylated Bases Methylated Cytosine " The 5 th base... " Cytosine 5-methyl Cytosine 64"
Purification WGBS Sequencing Analysis Detecting Methylated DNA Bisulfite Conversion 65"
DNA Methylation and Exercise " "Several striking new studies (show) that exercise seems able to drastically alter how genes operate" Reynolds (Times) " PLOS One study - Effect of 6-month exercise on genome-wide DNA methylation patterns. " Researchers found 39 candidate genes for obesity and type 2 diabetes that had differential DNA methylation in human adipose tissue after exercise. 66"
Purification WGBS Sequencing Analysis Historical WGBS Workflow Results: Yield Coverage Large amounts of DNA required but very low yield
Current Workflow Purify DNA Fragment (Covaris) End-Repair Ligate Adaptors BS Conversion PCR -Loss at each step -Multiple clean-ups -DNA Damage -Sample Loss -High PCR cycles Challenges: Covaris Lengthy protocol Loss due to DNA damage Methylated adapters Specialized proof reading pol 68"
Epicentre Solution EpiGnome Methyl-Seq Kit Purify DNA BS Conversion Random -Prime Tag PCR 50 ng input Post BS conversion library prep No Covaris No methylated adaptors No special polymerases Only 10c PCR
Purification WGBS Sequencing Analysis New: EpiGnome Methyl-Seq Kit Whole gnome bisulfite sequencing from only 50 ng gdna Results: Yield Coverage Bisulfite conversion happens first; EpiGnome captures ssdna for Full coverage of methylome No sample loss! 70"
Workflow in detail 71"
Purification WGBS Sequencing Analysis The Power of the Whole Genome CpG, CHG and CHH Methylation in a single data set Sensitive detection EpiGnome captures CpG, CHG & CHH methylation patterns Detect full spectrum of methylation
Purification WGBS Sequencing Analysis EpiGnome: Superior Performance EpiGnome NuGen ILMN/HB Input 50 ng 10-200 ng 1-5 ug BS Conversion Pre-Library Post-Library Post-Library Loss of BS fragments None ~90% ~90% Total Time 5 hrs 9 hrs 1-2 days PCR cycles 10 21 10+ Diversity 90%+?? 50-99% (DOI) Alignment >60%?? 30-80% Read length PE 75 100-200 PE 75/SR-50 Indexing Y (12-48) Y (16) N/Y EpiGnome No loss of coverage Standard primers (non-methylated) Standard Proof reading polymerase 73"
Illumina + Epicentre: Total Solution MasterPure EpiGnome White Paper 74"
Purification WGBS Sequencing Analysis Informatics Tools Begin with this: Continue with this: EpiGnome* Methyl Seq Bioinformatics User Guide Rev. 0.1 Introduction This guide contains data analysis recommendations for libraries prepared using EpicentreBs EpiGnome* Methyl Seq Kit, and sequenced on an Illumina sequencer. EpiGnome prepares whole genome bisulfite sequencing libraries (WGBS). The following test data set is available for download, and can be used as an example data set with this guide. The data set was generated using EpiGnome Methyl Seq Kit with 50 ng of Coriell gdna (GM12878) as input into bisulfite conversion. The libraries were sequenced with paired end 75 bp reads. The data set contains 10,000 reads from Read 1 and Read 2. http://www.epibio.com/wgbs_sample/sample_r1.fastq.gz http://www.epibio.com/wgbs_sample/sample_r2.fastq.gz IMPORTANT: This document provides information on data analysis for EpiGnome* Methyl Seq libraries that has been demonstrated internally and may be of interest to customers. The information is provided as is and is not an Epicentre product and is not accompanied by any rights or warranties. Customers using or adapting this information should obtain any licenses required and materials from authorized vendors. Epicentre products mentioned herein are for research use only unless marked otherwise. While customer feedback is welcomed, this user guide is not supported by Epicentre Technical Support or Field Application Scientists. 1 Korf Lab, UC Davis www.epibio.com 75"
Purification WGBS Sequencing Analysis Informatics EpiGnome Methyl Seq Kit Informatics workflow Sample Data at www.epibio.com 76"
The Illumina Value Chain: Total Workflow & Applications Solutions MasterPure" QuickExtract" TruSeq"&"ScriptSeq" TotalScript,"ART`Seq" Nextera" EpiGnome"(WGBS)" BaseSpace" Purifica:on% (rrna% Deple:on)% Library% Genera:on% Sequencing% Data% Analysis% Ribo`Zero" Globin`Zero" TS+Globin/RZ/PolyA" MiSeq,"HiSeq" Systems" 77"
Find the Best Tool for Your Project Molecular Biology Selection Guide Molecular Biology Selection Guide 78"
Find the Best Tool for Your Project Molecular Biology Selection Guide Quickly browse all available options for any project! 79"
80" Thank You!