Transcriptome sequencing in nematods

Presented at London Calling in London, England (sponsored by Oxford Nanopore Technologies)

Transcript

1. Transcriptome sequencing in nematodes Mallory Freeberg Postdoctoral Research Fellow, Johns Hopkins University May 26-27, 2016
2. © Copyright 2016 Oxford Nanopore Technologies ⎪ 2 ~3 days egg-to-lay Genetically tractable 100 Mb genome
3. © Copyright 2016 Oxford Nanopore Technologies ⎪ 3 C. elegans development proceeds through distinct stages Muscle Neuronal Intestinal Germline Hypodermal Embryonic
4. © Copyright 2016 Oxford Nanopore Technologies ⎪ 4 Developmental programs orchestrated by regulation at mRNA 3’UTRs hbl-1: hunchback-like, C2H2-type zinc finger TF Long 3’UTR (1392nt) let-7 miRNA binding sites Short 3’UTR (651nt) Expressed in embryo Expressed post-embryo Translational repression
5. © Copyright 2016 Oxford Nanopore Technologies ⎪ 5 1.  How can we achieve high-fidelity capture of 5’ and 3’ ends of polyadenylated, protein-coding transcripts for nanopore sequencing? 2.  Can nanopore reads provide robust identification of full-length transcripts, including alternative splicing events and 3’UTR isoforms? 3.  What can we learn about exon and 3’UTR isoform usage that regulates developmental programs and tissue-specific gene expression?
6. © Copyright 2016 Oxford Nanopore Technologies ⎪ 6 C. elegans L1 animals chosen for full-length mRNA seqeuncing
7. © Copyright 2016 Oxford Nanopore Technologies ⎪ 7 5’ and 3’ transcript ends captured by oligo(dT) priming and nicking Total RNA isolated from wild-type L1 animals 1st strand cDNA synthesized from total RNA using oligo(dT) primers   precise mRNA 3’ end capture   80-90% rRNA 2nd strand cDNA synthesized by nick translational replacement of RNA   3’-OH-primed synthesis   cDNA cleaned by spin column MinION library prepped according to standard protocols   No cDNA shearing -dT18
8. © Copyright 2016 Oxford Nanopore Technologies ⎪ 8 MinION run produced >77K high-quality 2D reads •  2D Basecalling for 26h •  Flowcell remuxed every 12h •  High-quality 2D reads aligned to C. elegans genome (ce10) with GraphMap1 2D read length (nt) No. 2D reads 0 4000 8000 High MAPQ (77%) Low MAPQ (14%) Unmapped (9%) Transcript length (nt) No. transcripts 0 4000 8000 12000 0 1000 3000 1. Sović et al. Nat Comm 2015 pass fail No. reads 0 50000 100000 2D read complement template 2D read complement template 77K pass fail No. bases 0.0e+00 1.0e+08 2.0e+08 2D read complement template 2D read complement template 140M
9. © Copyright 2016 Oxford Nanopore Technologies ⎪ 9 2D read length (nt) No. 2D reads 0 4000 8000 MinION read counts mirror expected transcript counts for L1 stage animals Transcript length (nt) Weighted transcript expr. 0 5000 15000 Transcript length (nt) No. transcripts 0 4000 8000 12000 0 1000 3000 2D read length (nt) No. 2D reads 0 4000 8000 High MAPQ (77%) Low MAPQ (14%) Unmapped (9%) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 0 50 100 150 0 50 100 150 Exp aggregate tx expr. Obs 2D read counts • 200nt bin Pearson correlation coefficient: 0.807 Spencer et al. Gen Res 2011 Observed Expected
10. © Copyright 2016 Oxford Nanopore Technologies ⎪ 10 5 kb ce10 7,479,000 7,480,000 7,481,000 7,482,000 7,483,000 7,484,000 7,485,000 7,486,000 7,487,000 7,4 34 MinION reads span full-length rod-1 locus Galaxy - Data Intensive Biology for Everyone rod-1 coding exons his-61 his-62 34 reads 6 partial reads
11. © Copyright 2016 Oxford Nanopore Technologies ⎪ 11 3’UTR cleavage orchestrated by polyadenylation signal (PAS) Canonical PAS (AAUAAA) Variant PAS PAS distance from 3’UTR end Number of 3’UTRs Magnone et al. Science 2009 3’ PAS 5’ cleavage site downstream signal mature 3’UTR
12. © Copyright 2016 Oxford Nanopore Technologies ⎪ 12 14-26nt down- stream of PAS2 14-26nt down- stream of PAS3 sense antisense 17 MinION 2D reads capture known rod-1 3’UTR isoforms PAS1 PAS2 PAS3 rod-1 3’UTR-short rod-1 3’UTR-long rod-1 exon
13. © Copyright 2016 Oxford Nanopore Technologies ⎪ 13 sense antisense 14-26nt down- stream of PAS2 14-26nt down- stream of PAS3 17 MinION 2D reads capture novel rod-1 3’UTR isoforms PAS1 PAS2 PAS3 rod-1 exon rod-1 3’UTR-short rod-1 3’UTR-long >130nt down- stream of PAS3
14. © Copyright 2016 Oxford Nanopore Technologies ⎪ 14 1.  How can we achieve high-fidelity capture of 5’ and 3’ ends of polyadenylated, protein-coding transcripts? Oligo(dT) priming (1st strand) + 3’-OH-priming (2nd strand) 2.  Do MinION reads provide robust identification of full-length transcripts, including alternative splicing events and 3’UTR isoforms?   Two know 3’UTRs identified for rod-1; potential novel 3’UTR 3.  What can we learn about exon and 3’UTR isoform usage that regulates developmental programs and tissue-specific gene expression?   Potential for identifying novel splicing and 3’UTR isoforms as single transcripts
15. © Copyright 2016 Oxford Nanopore Technologies ⎪ 15 Additional developmental stages including dauer and developing embryo Build better gene models for complicated alternatively spliced gens 10 kb ce10 14,650,000 14,655,000 14,660,000 14,665,000 unc-52: promotes muscle structure development 2 kb ce10 11,016,500 11,017,000 11,017,500 11,018,000 11,018,500 11,019,000 11,019,500 11,020,000 11,020,500 11,021,000 11,021,500 11,022,000 11,022,500 11,023,000 egl-15: fibroblast growth factor receptor PABP translating mRNA tag PABP *Pmyo-3 Tissue-specific exon and 3’UTR isoforms by pull-down of tissue-specific PABP *Pmyo-3: muscle-specific promoter
16. Acknowledgements: James Taylor, Ph.D. Michael Sauria, Ph.D. John Kim, Ph.D. Amelia Alessi, Ph.D.