Next generation sequencing Xusheng Wang 4/29/2010

Outline Background Technologies Data analysis and Applications

Why sequence DNA?

De novo sequencing genome Homo sapiens Mus musculus Rattus norvegicus Pan troglodytes Macaca mulatta Drosophila melanogaster Danio rerio Takifugu rubripes Arabidopsis thaliana oryza sativa Caenorhabditis elegans

Individual human genome sequencing

Interests to me… C57BL/6J (B) DBA/2J (D) F1 20 generations brother-sister matings BXD1 BXD2 BXD80 + … + F2 BXD RI Strain set BXD RI Strain set fully inbred fully inbred isogenic hetero- geneous hetero- geneous Recombined genomes are needed for mapping female male chromosome pair Inbred Isogenic siblings Inbred Isogenic siblings BXD

Cancer genome sequencing

Map-and-count experiments RNA-seqChIP-seq

History of DNA sequencing Messing & Llaca, PNAS (1998) Sanger Sequencer

Next generation sequencing technologies ABI SOLiDIllumina GA2 Roche 454

Single molecular sequencing technologies Helicos Single Molecule Real Time (SMRT) DNA sequencing

Comparison of NGS platforms Michael L. Metzker Nature Reviews Genetics 11, (Jan 2010)

Roche (454) GS FLX sequencer

Hiseq 2000

A A T T A A T T Prepare library Illumina Genome Analyzer

Prepare clusters Illumina Genome Analyzer

Prepare clusters Illumina Genome Analyzer

Sequencing Illumina Genome Analyzer A T C G

Sequencing Illumina Genome Analyzer A T C G

Sequencing Illumina Genome Analyzer A

Sequencing Illumina Genome Analyzer A T C G A

Sequencing Illumina Genome Analyzer A T

xwang TGGAAGAATATAGAGCCTGTCACAATCCTCCCTTTGAGCAGCATTAGTCTACAAAGGAAAAGAAAGT TCTCATGACTCTAGTGCCACCCTCACATACTTAC `_ab_ZaabbbaY`\_\a[[_a`aaa]`_aa\`aa[a\\aW]``a`VW \aa`aZ__Y]Z_aWZV_a][]][a`Y^X[\\[FKT``F\[^W`^TVZTVXODD xwang GGGCACTCTTGTGCGGCAACGGCTGGGTGAGGACTCAACGGGGCCCCGTCCTGTCTAGCCTCGC CCTCGCTTGCGGGACCAGACCGGACACTGGCGAAGTA X\\V_aa_aaa_a_R[[aa^U^`aV^_HXT[NMYPU_\PU]VTRZU[P K`_HIV[GG]IHSDG\_XYGPDW_LFHIOJT`ROJGTDDTZPIGVKJIJDGMD xwang CTTGCAGCAGATGTCTGGACTCCTCCAAAATACATGCCTAGGCGTCAACGCAGTTACCACCTGCTTT CCGCCAGTGATGCGTCCTCCTGGG.TCGCGTCTC VFHMMZOMZKMMZRGFFTHDZRFYMFHFDOTFJDDMEWKYHMMOHDDJ ZIDIGDDV]QIPNFGDODHHMFDDFDIFKDP_DHWDFDRHXHFDRGGGGHYDZ xwang ATGCTGACCAATCCGGAAC.CTCGGCTAGAAAACGCCAGGGGTCGAGAGAAGAATAATCTACAATCC GAAACAGCCAGGGAGTGAAACAGTATACGTGTAT [MRD[WKIPGSMJDQUVRDDJJPSDDMGPPYY_GGMDRFNFDDMHHDJ JHLPZMKDMOMJJDJDWIDRMNHIDHHDHLDNMDDRMMMDSKDDKHHNOFHDR 0 Data from Illumina GA2 LaneTileX Y Filter: 0-No; 1-Yes 1:Single; 2: paired endIndexMechine name

SOLiD system

SOLID sequencing

Ligation-based sequencing

Decoding color space Raw error rate = ~3% Corrected error rate = ~0.1%

Single SNP detection

Data from SOLiD (.csfasta) >4_27_99_F3 T >4_27_1062_F3 T >4_27_1570_F3 T >4_28_935_F3 T >4_28_1306_F3 T >4_29_429_F3 T >4_29_506_F3 T >4_29_636_F3 T >4_29_940_F3 T >4_29_1957_F3 T >4_31_522_F3 T >4_31_1523_F3 T

Quality value (_QV.qual) >4_27_99_F >4_27_1062_F >4_27_1570_F >4_28_935_F >4_28_1306_F >4_29_429_F

Comparing Sequencers read length bases per machine run 10 bp1,000 bp200 bp 1 Gb 100 Mb 1Mb 100Gb Illumina, AB/SOLiD short-read sequencers ABI capillary sequencer 454 pyrosequencer ( Mb in bp reads) (100Gb in bp reads)

Comparing Sequencers Roche (454)Illumina GASOLiD ChemistryPyrosequencingReverse terminatorLigation-based AmplificationEmulsion PCRBridge AmpEmulsion PCR Paired ends/sepYes/3kbYes/200 bpYes/3 kb Mb/run100 Mb20 Gb100 Gb Time/run7 h4 days/8days7 days / 14 days Read length400 bp bp50 bp

Data volume

… and they give you the picture on the box Read mapping Read mapping is like doing a jigsaw puzzle… …you get the pieces… Problem is, some pieces are easier to place than others…

Alignment of reads Reads generated from sequencing is mapped to a reference genome Conventional tools like Blast or Blat do not work well with short sequence reads. Modification of existing alignment algorithms to handle short reads.

Alignment Tools ELAND MAQ Bowtie SOAP Bioscope/Corona Lite pipeline Tophat BFAST

SAM/BAM format File format version Sequence name; Sequence length read group

Sequence variations SNPsInsertionDeletion Medium Insertion

SNPs between the C57BL/6J and DBA/2J 4,553,000 SOLiDIllumina

Evaluating SNPs calls 5% adjacent SNPs 20% adjacent SNPs

21 August 2015 D 321 ChrID Supports: TAAGAATGAGTTGGCAAATAAAGAGTTTGGTGAGTTTATAGAAATATAGGggccg ataggACAAGGTACAAGGAATGGCTGAAGGAGAGAGGTTG GAGTTTATAGAAATATAGG ACAAGGTACAAGGAATG GTGAGTTTATAGAAATATAGG ACAAGGTACAAGGAA GAGTTTATAGAAATATAGG ACAAGGTACAAGGAATG TGGTGAGTTTATAGAAATATAGG ACAAGGTACAAGG GAGTTTATAGAAATATAGG ACAAGGTACAAGGAATG AGTTTGGTGAGTTTATAGAAATATAGG ACAAGGTACAAGGA GTGAGTTTATAGAAATATAGG ACAAGGTACAAGGAA AGTTTATAGAAATATAGG ACAAGGTACAAGGAATGG TTTGGTGAGTTTATAGAAATATAGG ACAAGGTACAA TGAGTTTATAGAAATATAGG ACAAGGTACAAGGAATG TGAGTTTATAGAAATATAGG ACAAGGTACAAGGAAT GTTTATAGAAATATAGG ACAAGGTACAAGGAATGGC GAGTTTATAGAAATATAGG ACAAGGTACAAGGAATG AGTTTATAGAAATATAGG ACAAGGTACAAGGAATGG base - 1million bases Medium indels detection

Large indels detection K. Chen et al., Nature Methods 6: (2009) Concordance Insertion Deletion Clone inserted size

InDels between the C57BL/6J and DBA/2J

Inversion detected by paired-end data Total Inversions Span exon(s) or gene(s) IntronsIntergenic

Copy Number Variations (CNVs) Total CNVsGainsLosses 21,7397,18214,557 Graubert, et al PLoS Genetics Anderson, et al Genes & Immunity Several gene members of Klra family was deleted in DBA/2J

De novo assembly ABySS ALLPATHS Euler-SR SHRAP SSAKE Velvet SOAP

Variation viewed at a genome scale

RNA sequencing

.csfasta Filtering Ribosomal RNA tRNA TTTT / AAAA Adapters Alignments Reference sequences Merging and sorting Counting reads Novel transcripts RNA sequencing analysis pipeline

Alignment methods Transcriptome reads that cross splice junctions Anchor Extend method

Alternative splicing Novel Transcribed Region (NTR) Definition: a segment of genomic sequence that is transcribed but is not currently annotated as an exon in a database

Our RNAseq data on UCSC genome browser bin/hgTracks?hgS_doOtherUser=submit&hgS_otherUserName=Williamslab&hgS_otherUserSessionName=eye _RNAseq

Finding the new SNP data

Finding the indel data

Using the new sequence data