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Full-length cDNA Sequencing.pdf
- 1. FIND MEANING INCOMPLEXITY © Copyright 2013 by Pacific Biosciences of California, Inc. All rights reserved. Profiling Full-Length cDNAs by SMRT® Sequencing
- 2. Short Reads FallShort in the Era of “Alternative Events” 2 Alternative transcription start sites Alternative splicing Alternative polyA sites AAAA AAAA Genome Transcriptome Transcriptome Transcriptome Transcriptome Transcriptome Transcriptome Transcriptome Transcriptome Transcriptome
- 3. Bringing Full-Length cDNASequencing to the PacBio® RS PacBio read lengths offer a unique opportunity for transcriptome biology Methods development/optimization • Robust synthesis of full-length cDNA libraries • Sample normalization • Target enrichment • Sequencing recommendations • Paths to analysis 3 Cumulative distribution of human transcripts in 2 databases Length (nucleotides) Au et al. PLoS ONE, 2012
- 4. Full-Length cDNA SynthesisKit - Invitrogen 4 • Very time-consuming • Large polyA RNA input • More stringent about full length cDNA because of cap selection • 5 μg of polyA RNA • 50-100 ng of ds cDNA • Successfully scaled down the input to 1 μg polyA RNA
- 5. “Full-Length” cDNA: TemplateSwitching Matz M, Shagin D, Bogdanova E, Britanova O, Lukyanov S, Diatchenko L, Chenchik A (1999) Amplification of cDNA ends based on template-switching effect and step-out PCR. Nucleic Acids Res. 27, 1558-1560. 5 • Clontech® SMART kit • Evrogen® Mint-2 kit • Uses less material • Less time consuming/fewer steps • Less stringent about selecting full-length cDNA
- 6. cDNA SMRTbell™ LibrariesDisplay Size Distributions That Correlate with Expected Full-Length mRNA Sizes 6 S. cerevisiae H. Sapiens cerebellum 500 5k 500 5k cDNA synthesis method PCR PacBio SMRTbell™ library prep
- 7. Both Kits ProduceGood Results with a High Quality Sample 7 Clontech® Human Cerebellum polyA RNA Aligned to Gencode by BLASR 5’ 3’ 5’ 3’ Invitrogen Evrogen More 3’ bias
- 8. Lower Quality RNABenefits from the 7mG Enrichment used in Invitrogen® Method 8 MAQC-B (Human brain cDNA) 1-2 kb cDNA fraction Clontech Invitrogen
- 9. Normalization During cDNASample Preparation 9 1 μg input 4-7 hr procedure Unknown yield into PCR PCR of 15-20 cycles to get DNA for SMRTbell™ template prep Zhulidov PA et al. A method for the preparation of normalized cDNA libraries enriched with full-length sequences. Bioorg Khim. 2005; 31 (2):186-94. Zhulidov PA et al. Simple cDNA normalization using kamchatka crab duplex- specific nuclease. Nucleic Acids Res. 2004; 32 (3):e37.
- 10. Normalization Increases theSequence Breadth of a Sample 10 Normalized Non normalized Yeast Full-Length cDNA aligned to genome by BLASR Coverage Chr IV Coverage Chr VII
- 11. Normalization Increases theSequence Breadth of a Sample 11 3485 1736 Normalized Non-Normalized 306 Number of Genes observed in a single SMRT® Cell of transcript data
- 12. Subread Length Distribution H1 humanstem cell cDNA: Clontech No size selection SMRTbell™ library size: Bioanalyzer Improving Subread Read Lengths for cDNA Libraries 12 500 5k
- 13. 13 H1 SMRTbell™ librarysize: Bioanalyzer Subread Length Distribution 1-2 kb fraction 2-3 kb fraction >3 kb fraction Improving Subread Lengths for cDNA Libraries
- 14. Agilent® SureSelect® Enrichmentto Increase the Coverage of a Subset of cDNAs 14 Full-Length ds_cDNA Enriched Full-Length ds_cDNA
- 15. Agilent® Kinome EnrichmentSignificantly Increases Target Reads 15 Precision = # reads / total reads 0.015 0.61
- 16. Detection of NovelSplice Forms of a Cyclin-Dependent Kinase 16
- 17. Shared Protocol Availableon Sample Net http://www.smrtcommunity.com/Share/Protocol?id=a1q70000000 H6LmAAK&strRecordTypeName=Protocol 17
- 18. Analysis Options: TranscriptomeReference • If alignment to a transcript database is desired, BLASR and BWA-SW can be used for alignment 18 RefSeq/Gencode BLASR • https://github.com/PacificBiosciences/blasr BWA-SW • http://bio-bwa.sourceforge.net/
- 19. Analysis Options: GenomicReference-Based Alignment • BLAT and GMAP can be used to align PacBio CLR and CCS reads to the genomic reference. 19 Genomic Reference BLAT • http://www.soe.ucsc.edu/~kent/src GMAP • http://research-pub.gene.com/gmap/
- 20. Short Reads andGenomic-Reference-Based Alignment • If short-read data is available, error correction can be done prior to alignment. 20 Short Read / CCS Data pacBioToCA P_ErrorCorrection LSC Genomic Reference BLAT GMAP
- 21. cDNA Takeaways Sample PrepRun Design Sequencing on the PacBio® RS and primary analysis Secondary Analysis Tertiary Analysis • Two cDNA prep methods show promising results but differ in input and stringency • Double-stranded cDNA is converted to SMRTbell™ libraries with the PacBio® Large Insert Kit • Normalization can be an effective means to increase breadth, but use caution if characterizing rare isoforms • Agilent® SureSelect® system is effective for custom enrichment of select genes • Size Selection Can Enrich for Larger cDNAs • Run design depends on size • <2k fractions: 2x45 or 2x55 min movies, C2/C2, diffusion loading, CPS start • >2k fractions: 1x120 min movies, mag loading, XL/C2, stage start • Non-size selected: run both conditions above to cover all sizes, or select for desired size range • Full-Pass Subreads represent putative full-length Isoforms • Transcript Reference • BLASR • BWA-SW • Genomic Reference • BLAT • GMAP • Error Correction: Short Reads • pacBioToCA • P_ErrorCorrection • LSC (Au et al. PLoS ONE, 2012)