Going Beyond SNPs with Next Genera5on Sequencing Technology Personalized Medicine: Understanding Your Own Genome Fall 2014

Transcription

1 Going Beyond SNPs with Next Genera5on Sequencing Technology Personalized Medicine: Understanding Your Own Genome Fall 2014

2 Next Genera5on Sequencing Technology (NGS) NGS technology Discover more genefc polymorphisms by sequencing genomes of many individuals More importantly, NGS technology allows us to discover structural variants! Other applicafons of NGS technology RNA- Seq: sequencing transcriptome to measure mrna expression levels ChIP- Seq: sequencing transcripfon factor (TF) binding sites on DNA to study TF- target interacfons

3 1 Kb to several Mb in size Genomic Rearrangements/ Structural Varia5ons (SVs) courtesy of Tobias Rausch (EMBL)

4 1 Kb to several Mb in size Copy number variants (CNVs) DeleFon DuplicaFon Genomic Rearrangements/ Structural Varia5ons (SVs) courtesy of Tobias Rausch (EMBL)

5 1 Kb to several Mb in size Copy number variants (CNVs) DeleFon DuplicaFon InserFon Genomic Rearrangements/ Structural Varia5ons (SVs) courtesy of Tobias Rausch (EMBL)

6 1 Kb to several Mb in size Copy number variants (CNVs) DeleFon DuplicaFon InserFon, Inversion Genomic Rearrangements/ Structural Varia5ons (SVs) courtesy of Tobias Rausch (EMBL)

7 Genomic Rearrangements/ Structural Varia5ons 1 Kb to several Mb in size Copy number variants DeleFon DuplicaFon InserFon, Inversion, TranslocaFon Either neutral or non- neutral in funcfon Non- neutral mechanisms DisrupFng genes CreaFng fusion genes Copy number changes of dosage- sensifve genes courtesy of Tobias Rausch (EMBL)

8 DNA sequencing vectors DNA Shake DNA fragments Vector Circular genome (bacterium, plasmid) + = Known location (restriction site) Adopted from hzp://

9 Method to sequence longer regions genomic segment cut many times at random (Shotgun) Get two reads from each segment ~500 bp ~500 bp Adopted from hzp://

10 ReconstrucFng the Sequence (Fragment Assembly) reads Cover region with ~7-fold redundancy (7X) Overlap reads and extend to reconstruct the original genomic region Adopted from hzp://

11 Defini5on of Coverage C Length of genomic segment: Number of reads: n Length of each read: l L Defini5on: Coverage C = n l / L Adopted from hzp://

12 Depth of Coverage and Physical Coverage Single- end sequencing Paired- end sequencing Paired- end sequencing

13 Compu5onal Methods for Detec5ng Genomic Rearrangements Reference Mate-pair or paired-end mapping abnormalities Split-Read alignments Read depth signals courtesy of Tobias Rausch (EMBL)

14 Paired-end data Next- GeneraFon Sequencing (NGS) for detecfng structural variafons Mate pair A pair of sequenced reads that came from each end of the same DNA fragment

15 Insert Size for a Mate Pair For each fragment Sequenced read Insert size Sequenced read Let s assume we know the insert size for now.

16 courtesy of Tobias Rausch (EMBL)

17 courtesy of Tobias Rausch (EMBL)

18 Insert Size for a Mate Pair For each fragment Sequenced read Insert size Sequenced read In pracfce, do we know the insert size?

19 Paired-end data paired-end NGS (insert distribution known due to fragment size selection)

20 Insertions! " Deletions courtesy of Tobias Rausch (EMBL)

21 courtesy of Tobias Rausch (EMBL)

22 courtesy of Tobias Rausch (EMBL)

23 courtesy of Tobias Rausch (EMBL)

24 courtesy of Tobias Rausch (EMBL)

25 courtesy of Tobias Rausch (EMBL)

26 courtesy of Tobias Rausch (EMBL)

27 1 Copy 1 Copy 0 Copy 2 Copy 2 Copy courtesy of Tobias Rausch (EMBL) # Chiang et al. (2009)

28 Down- Syndrom ParFal Trisomie 21 courtesy of Tobias Rausch (EMBL) # Xie et al. (2009)

29 With reads of length bps are we able to find the exact breakpoint of a structural variafon?

30 With reads of length bps are we able to find the exact breakpoint of a structural variafon? Yes using split- read mapping Donor Reference

31 With reads of length bps are we able to find the exact breakpoint of a structural variafon? Yes using anchored split- read mapping Donor Reference mappable read mate provides anchor to narrow down search space # Medvedev et al. (2009)

32 The Pindel algorithm (Dele5ons) # Ye et al. (2009)

33 The Pindel algorithm (Real Data) # Ye et al. (2009)

34 Detec5ng Gene5c Polymorphisms from NGS Reads

35 Detec5ng Gene5c Polymorphisms from NGS Reads

36 Next Genera5on Sequencing Technology (NGS) NGS technology Discover more genefc polymorphisms by sequencing genomes of many individuals More importantly, NGS technology allows us to discover structural variants! Other applicafons of NGS technology RNA- Seq: sequencing transcriptome to measure mrna expression levels ChIP- Seq: sequencing transcripfon factor (TF) binding sites on DNA to study TF- target interacfons

37 RNA- Seq Massively parallel sequencing method for transcriptome analyses Complementary DNA (cdna) generated from RNA are sequenced using next- generafon short read technologies Reads are aligned to a reference genome and a transcriptome map is constructed

38 Next Genera5on Sequencing (NGS) based methods RNA- Seq can determine both mrna abundance and sequence content Advantages over microarrays Rare transcripts discovery AlternaFve splicing event detecfon Transcript sequence variafon detecfon

39 mrna- Seq vs microarrays Sultan, Schulz, Richard et. al 2008

40 mrna- Seq vs microarrays (2) Common to arrays and mrna- Seq Only Detected by mrna- Seq Sultan, Schulz, Richard et. al 2008

41 Microarrays vs. RNA- seq: Pros and Cons Microarrays Well established technologies Cheap Inaccurate You may only find what you did design RNA-Seq Captures abundance and sequence information Accurate Sensitive to lowly expressed transcripts Can detect alternative splicing More expensive than arrays Huge data amounts create analysis challenges Read alignment can be difficult

42 Challenges of Read Alignment for RNA- Seq Currently, our knowledge of the transcriptome is incomplete No direct mapping of reads to the transcriptome, but needs to use the reference genome as a proxy AlternaFve splicing Some of the reads may span exon juncfons and may not directly align to the reference genome confguously For known exons and isoforms, the reads can be aligned to concatenated exons For rarely transcribed genes, the exon juncfons may be spanned by only few reads

43 ChIP- Seq ChIP- Seq: methods for measuring genome- wide profiles of immunoprecipitated DNA- protein complexes ChIP- Seq can be used to detect transcripfon factor binding sites on DNA

44 Summary Next generafon sequencing technology Understand coverage Single- end vs paired- end sequencing DetecFng structural variafon with NGS Paired- end sequencing: examine mapped distances to the reference genome Single- end sequencing: examine read depth Other applicafons of NGS: RNA seq, ChIP seq