Applied Biosystems SOLiD 3 Plus System. RNA Application Guide

Transcription

1 Applied Biosystems SOLiD 3 Plus System RNA Application Guide

2 For Research Use Use Only. Not intended for any animal or human therapeutic or diagnostic use. TRADEMARKS: Trademarks of Life Technologies Corporation and its affiliated companies: AB (Design), AB Logo, Applied Biosystems, BioScope, MagMAX, mirvana, Poly(A)Purist, RiboMinus, RNAqueos, SOLiD. Trizol is a registered trademark of Molecular Research Center, Inc. Sage is a trademark of Genzyme Corporation. All other trademarks are the sole property of their respective owners. Copyright 2010, Life Technologies Corporation. All rights reserved. 2/2010

3 Contents Summary of RNA applications Whole transcriptome analysis Small RNA analysis SOLiD SAGE analysis DRAFT February 12, :07 am, RNA Application GuideTOC.fm i

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5 Summary of RNA applications Summary of RNA applications Paramter Whole Transcriptome Small RNA SAGE Discovery or Quantitation Discovery & Quantitation of known RefSeqs and non-coding RNAs Quantitation of known RefSeqs Discovery & Quantitation of known small RNA & isoforms Quantitation of known small RNA Quantitation of known RefSeqs Input RNA 200 ng rrna depleted RNA 100 ng polya RNA 20 ng bp size selected RNA 50 ng polya RNA Read Length 50 bp 50 bp 35 bp 35 bp 35 bp Recommended Number of Reads Novel Transcripts >100 M 50 M mappable up to 30 M mappable mappable Yes 5 M mappable 2-3 M mappable Novel Exons Yes NA NA Novel Junctions Yes NA NA DRAFT csnps Yes NA NA Low Exp Trans. Software Third Party Software Yes BioScope rna2map SOLiD SAGE Analysis Software Yes # As per published product protocols for SOLiD Whole Transcriptome Kit, SOLiD RNA Expression Kit and SOLiD SAGE Kit. Read estimates based on human genome; smaller or less complex genomes are estimated to require proportionally fewer reads. The mappability will be driven by quality of reference and user circumstances. All mapping percentages are estimates based on existing analysis tools and reference samples. These numbers are expected to increase as analysis tools and annotations improve. # SOLiD Software Community Commercial Vendor Solutions at info.appliedbiosystems.com/solidsoftwarecommunity 1

6 2 Whole transcriptome analysis Sample Sample preparation starts with the isolation of RNA. The type of RNA used as input is dictated by the question at hand (total RNA, poly A+ RNA, rrna depleted RNA). RNA quality and quantity play an important role in the success of experiments. Next the RNA is used to create a fragment library. Emulsion PCR is used to make templated beads. The beads are deposited onto slide for sequencing on the SOLiD Analyzer using the appropriate chemistry. Templated bead Production Input Data Analysis BioScope WT Pipeline Output Whole transcriptome analysis MagMAX Total RNA Isolation Kits RNAqueous Kits Poly(A)Purist mrna Purification Kits Whole Transcriptome Analysis Kit Transcriptome Multiplexing Kit RiboMinus Kits for RNA- Seq epcr Kit V2 Buffer Kit Bead Enrichment Kit Bead Deposition Kit Slide Pack Kit Opti Fragment Master Mix 50 Opti Fragment Kit- 5 bp Barcode Set Instrument Buffer Kit Workflow Analysis Reagents Data from run are exported off instrument for analysis (automated).csfasta &.qual file formats Data is auto analyzed via Bioscope software s Whole Transcriptome Pipeline Map reads to genome Counts reads for genes or exons Third Party Software Mapping data provided in base space with base QV and mapping QV (continuous mapped reads), enabling mapping to known and putative splice junctions (SAM/BAM file formats) Count of known exons counts are provided for all the exons that are mapped to the reference (gff and gtf file formats, respectively) Viewing each chromosome will have files for positive and negative Mapping can also be carried out using the academic software RNA-MATE, information available at Other commercial software for analysis of Whole Transcriptome data is available from Geospiza, GenomeQuest, SoftGenetics and Partek. information available at info.appliedbiosystems.com/solidsoftwarecommunity

7 Small RNA analysis Sample mirvana mirna Isolation Kits Sample preparation starts with the isolation of RNA. Note that RNA quality and quantity play an important role in the success of experiments. Next a fragment library is created. Emulsion PCR is used to make templated beads. The beads are deposited onto slide for sequencing on the SOLiD Analyzer using the appropriate chemistry. Small RNA Expression Kit Templated bead Production epcr Kit V2 Buffer Kit Bead Enrichment Kit Bead Deposition Kit Slide Pack Kit Opti Fragment Master Mix 35 Opti Fragment Kit- 5 bp Barcode Set Instrument Buffer Kit Workflow Analysis Reagents Input Data from run are exported off instrument for analysis (automated).csfasta &.qual file formats Data Analysis Small RNA Pipeline Data is analyzed via SOLiD System Small RNA Analysis Tool Map data sequentially to the filter reference, mirbase and genome Count reads for annotated regions of mirbase. Output Mapping data reads which map to the filter, mirbase and genome references will be provided (.ma file format) Counts of small RNAs reads counts of reads that match annotated regions of mirbase reference (gff, strand specific) Viewing files for both the reads mapped to the mirbase and the genome (positive and negative stand) for viewing UCSC genome browser (wiggle file format) Third Party Software Mapping can also be carried out using the academic software SHRiMP, information available at Other commercial software for analysis of small RNA data are available from Geospiza, InterRNA, and Partek, information available at info.appliedbiosystems.com/solidsoftwarecommunityty Small RNA analysis 3

8 4 SOLiD SAGE analysis Sample Sample preparation starts with the isolation of RNA. Note that RNA quality and quantity play an important role in the success of experiments. Next a fragment library is created. Emulsion PCR is used to make templated beads. The beads are deposited onto slide for sequencing on the SOLiD Analyzer using the appropriate chemistry. Trizol Reagent SAGE kit Templated bead Production epcr Kit V2 Buffer Kit Bead Enrichment Kit Bead Deposition Kit Slide Pack Kit Opti Fragment Master Mix 35 Instrument Buffer Kit Workflow Analysis Reagents Input Data from run are exported off instrument for analysis (automated).csfasta &.qual file formats Data Analysis SOLiD SAGE Analysis Software Data is analyzed via SOLiD SAGE Analysis Software Map the data to the RefSeq (sense & antisense) and genome. Count the number reads for each tag May include comparison of samples Third Party Software Output Mapping & Counting- A tab delimited text file (TDT) lists each tag sequence, its frequency of occurrence, its GenBank Identifier (GI) number, and a description of the identified gene Comparison - A tab delimited text file (TDT) comparing the tags in two different samples. Other commercial software for analysis of SOLiD SAGE data are available from SoftGenetics, Geospiza, GenomeQuest, and Partek, information available at info.appliedbiosystems.com/solidsoftwarecommunity SOLiD SAGE analysis

9 SOLiD SAGE analysis DRAFT 5

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