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Analysing re-sequencing samples Anna Johansson Anna.johansson@scilifelab.se WABI / SciLifeLab Re-sequencing Reference genome assembly ...GTGCGTAGACTGCTAGATCGAAGA... Re-sequencing IND 1 IND 2 IND 3 IND 4 GTAGACT TGCGTAG TAGACTG

  1. Analysing re-sequencing samples Anna Johansson Anna.johansson@scilifelab.se WABI / SciLifeLab

  2. Re-sequencing Reference genome assembly ...GTGCGTAGACTGCTAGATCGAAGA...

  3. Re-sequencing IND 1 IND 2 IND 3 IND 4 GTAGACT TGCGTAG TAGACTG AGATCGA AGATCGG ATCGAAG GATCGAA GTAGACT GCGTAGT AGACTGC GACTGCT GATCGAA Reference genome assembly ...GTGCGTAGACTGCTAGATCGAAGA...

  4. Re-sequencing IND 1 IND 2 IND 3 IND 4 GTAGACT TGCGTAG TAGACTG AGATCGA AGATCGG ATCGAAG GATCGAA GTAGACT GCGTAGT AGACTGC GACTGCT GATCGAA Reference genome assembly ...GTGCGTAGACTGCTAGATCGAAGA...

  5. Re-sequencing IND 1 IND 2 IND 3 IND 4 GTAGACT TGCGTAG TAGACTG AGATCGA AGATCGG ATCGAAG GATCGAA GTAGACT GCGTAGT AGACTGC GACTGCT GATCGAA Reference genome assembly ...GTGCGTAGACTGCTAGATCGAAGA... IND 1 IND 2 IND 3 IND 4 5 GTAGACT 16 TGCGTAG 24 AGTTCGA 8 AGATCGA 12 AGTTCGG 6 ATCGAAG 5 GTAGACT 19 GTAGGCT 3 GCGTAGT 7 AAACTGC 18 GATCGAA 2 GATCGAA

  6. Rare variants in human

  7. Exome sequencing in trios to detect de novo coding variants

  8. Population genetics – speciation, adaptive evolution Darwin Finches

  9. Population genetics – speciation, adaptive evolution Darwin Finches Heliconius Butterflies

  10. Population genetics – speciation, adaptive evolution Darwin Finches Heliconius Butterflies Lake Victoria cechlid fishes

  11. Paired end sequencing

  12. Pair-end reads • Two .fastq files containing the reads are created • The order in the files are identical and naming of reads are the same with the exception of the end • The naming of reads is changing and depends on software version ID_R1_001.fastq ID_R2_001.fastq @HISEQ:100:C3MG8ACXX: @HISEQ:100:C3MG8ACXX: 5:1101:1160:2197 1:N:0:ATCACG 5:1101:1160:2197 2:N:0:ATCACG CAGTTGCGATGAGAGCGTTGAGAAGTATAATAGG CTTCGTCCACTTTCATTATTCCTTTCATACATG AGTTAAACTGAGTAACAGGATAAGAAATAGTGAG CTCTCCGGTTTAGGGTACTCTTGACCTGGCCTT ATATGGAAACGTTGTGGTCTGAAAGAAGATGT TTTTCAAGACGTCCCTGACTTGATCTTGAAACG + + B@CFFFFFHHHHHGJJJJJJJJJJJFHHIIIIJJ CCCFFFFFHHHHHJJJJIJJJJJJJJJJJJJJJ JIHGIIJJJJIJIJIJJJJIIJJJJJIIEIHHIJ JJJJJJJIJIJGIJHBGHHIIIJIJJJJJJJJI HGHHHHHDFFFEDDDDDCDDDCDDDDDDDCDC JJJHFFFFFFDDDDDDDDDDDDDDDEDCCDDDD

  13. Pair-end reads • Two .fastq files containing the reads are created • The order in the files are identical and naming of reads are the same with the exception of the end • The naming of reads is changing and depends on software version ID_R1_001.fastq ID_R2_001.fastq @HISEQ:100:C3MG8ACXX: @HISEQ:100:C3MG8ACXX: 5:1101:1160:2197 1:N:0:ATCACG 5:1101:1160:2197 2:N:0:ATCACG CAGTTGCGATGAGAGCGTTGAGAAGTATAATAGG CTTCGTCCACTTTCATTATTCCTTTCATACATG AGTTAAACTGAGTAACAGGATAAGAAATAGTGAG CTCTCCGGTTTAGGGTACTCTTGACCTGGCCTT ATATGGAAACGTTGTGGTCTGAAAGAAGATGT TTTTCAAGACGTCCCTGACTTGATCTTGAAACG + + B@CFFFFFHHHHHGJJJJJJJJJJJFHHIIIIJJ CCCFFFFFHHHHHJJJJIJJJJJJJJJJJJJJJ JIHGIIJJJJIJIJIJJJJIIJJJJJIIEIHHIJ JJJJJJJIJIJGIJHBGHHIIIJIJJJJJJJJI HGHHHHHDFFFEDDDDDCDDDCDDDDDDDCDC JJJHFFFFFFDDDDDDDDDDDDDDDEDCCDDDD

  14. Mapping of pair-end reads Insert size

  15. Adaptor trimming module add cutadapt 3' Adapter When the adaptor has been read in sequencing it is present in reads or and needs to be removed prior to mapping 5' Adapter or Read Anchored 5' adapter Adapter Removed sequence

  16. Basic quality control - FASTQC module add FastQC

  17. Steps in resequencing analysis 1) Setup programs, data 2,3,4) map reads to a reference find best placement of reads bam file realign indels remove duplicates 5) recalibrate alignments recalibrate base quality bam file statistical algorithms 6) identify/call variants to detect true variants vcf file

  18. GATK version

  19. When in doubt, google it!

  20. Steps in resequencing analysis 1) Setup programs, data 2,3,4) map reads to a reference find best placement of reads bam file realign indels remove duplicates 5) recalibrate alignments recalibrate base quality bam file statistical algorithms 6) identify/call variants to detect true variants vcf file

  21. brute force TCGATCC x GACCTCATCGATCCCACTG

  22. brute force TCGATCC x GACCTCATCGATCCCACTG

  23. brute force TCGATCC x GACCTCATCGATCCCACTG

  24. brute force TCGATCC x GACCTCATCGATCCCACTG

  25. brute force TCGATCC ||x GACCTCATCGATCCCACTG

  26. brute force TCGATCC x GACCTCATCGATCCCACTG

  27. brute force TCGATCC x GACCTCATCGATCCCACTG

  28. brute force TCGATCC ||||||| GACCTCATCGATCCCACTG

  29. hash tables build an index of the reference sequence for fast access 0 5 10 15 GACCTCATCGATCCCACTG seed length 7 à chromosome 1, pos 0 GACCTCA à chromosome 1, pos 1 ACCTCAT à chromosome 1, pos 2 CCTCATC à chromosome 1, pos 3 CTCATCG à chromosome 1, pos 4 TCATCGA à chromosome 1, pos 5 CATCGAT à chromosome 1, pos 6 ATCGATC à chromosome 1, pos 7 TCGATCC à chromosome 1, pos 8 CGATCCC à chromosome 1, pos 9 GATCCCA

  30. hash tables build an index of the reference sequence for fast access TCGATCC ? 0 5 10 15 GACCTCATCGATCCCACTG à chromosome 1, pos 0 GACCTCA à chromosome 1, pos 1 ACCTCAT à chromosome 1, pos 2 CCTCATC à chromosome 1, pos 3 CTCATCG à chromosome 1, pos 4 TCATCGA à chromosome 1, pos 5 CATCGAT à chromosome 1, pos 6 ATCGATC à chromosome 1, pos 7 TCGATCC à chromosome 1, pos 8 CGATCCC à chromosome 1, pos 9 GATCCCA

  31. hash tables build an index of the reference sequence for fast access TCGATCC = chromosome 1, pos 7 0 5 10 15 GACCTCATCGATCCCACTG à chromosome 1, pos 0 GACCTCA à chromosome 1, pos 1 ACCTCAT à chromosome 1, pos 2 CCTCATC à chromosome 1, pos 3 CTCATCG à chromosome 1, pos 4 TCATCGA à chromosome 1, pos 5 CATCGAT à chromosome 1, pos 6 ATCGATC à chromosome 1, pos 7 TCGATCC à chromosome 1, pos 8 CGATCCC à chromosome 1, pos 9 GATCCCA

  32. Burroughs-Wheeler Aligner algorithm used in computer science for file compression original sequence can be reconstructed BWA ( module add bwa ) Burroughs-Wheeler Aligner

  33. Input to mapping – reference + raw reads Reference genome assembly Ind .fasta + fasta.fai R1.fastq R2.fastq >Potra000002 @HISEQ:100:C3MG8ACXX:5:1101:1160:2197 1:N:0:ATCACG CACGAGGTTTCATCATGGACTTGGCACCAT CAGTTGCGATGAGAGCGTTGAGAAGTATAATAGGAGTTAAACTGAGTAACAGG AAAAGTTCTCTTTCATTATATTCCCTTTAG ATAAGAAATAGTGAGATATGGAAACGTTGTGGTCTGAAAGAAGATGT GTAAAATGATTCTCGTTCATTTGATAATTT + TGTAATAACCGGCCTCATTCAACCCATGAT B@CFFFFFHHHHHGJJJJJJJJJJJFHHIIIIJJJIHGIIJJJJIJIJIJJJJ CCGACTTGATGGTGAATACTTGTGTAATAA IIJJJJJIIEIHHIJHGHHHHHDFFFEDDDDDCDDDCDDDDDDDCDC CTGATAATTTACTGTGATTTATATAACTAT @HISEQ:100:C3MG8ACXX:5:1101:1448:2164 1:N:0:ATCACG CTCATAATGGTTCGTCAAAATCTTTTAAAA NAGATTGTTTGTGTGCCTAAATAAATAAATAAATAAAAATGATGATGGTCTTA GATAAAAAAAACCTTTATCAATTATCTATA AAGGAATTTGAAATTAAGATTGAGATATTGAAAAAGCAGATGTGGTC TAAATTCAAATTTGTACACATTTACTAGAA + ATTACAACTCAGCAATAAAATTGACAAAAT #1=DDFFEHHDFHHJGGIJJJJGIHIGIJJJJJIIJJJJIJJJFIJJF? ATAAAACAGAACCGTTAAATAAGCTATTAT FHHHIIJJIIJJIGIIJJJIJIGHGHIIJJIHGHGHGHFFFEDEEE>CDDD TTATTTCATCACAAAACATCTAAGTCAAAA @HISEQ:100:C3MG8ACXX:5:1101:1566:2135 1:N:0:ATCACG ATTTGACATAAGTTTCATCAATTTACAAAC NTATTTTTGCTATGTGTCTTTTCGTTTTAAGTCTCCTTGTTGATATTTTTACA

  34. Output from mapping - SAM format HEADER SECTION @HD VN:1.0 SO:coordinate @SQ SN:1 LN:249250621 AS:NCBI37 UR:file:/data/local/ref/GATK/human_g1k_v37.fasta M5:1b22b98cdeb4a9304cb5d48026a85128 @SQ SN:2 LN:243199373 AS:NCBI37 UR:file:/data/local/ref/GATK/human_g1k_v37.fasta M5:a0d9851da00400dec1098a9255ac712e @SQ SN:3 LN:198022430 AS:NCBI37 UR:file:/data/local/ref/GATK/human_g1k_v37.fasta M5:fdfd811849cc2fadebc929bb925902e5 @RG ID:UM0098:1 PL:ILLUMINA PU:HWUSI-EAS1707-615LHAAXX-L001 LB:80 DT:2010-05-05T20:00:00-0400 SM:SD37743 CN:UMCORE @RG ID:UM0098:2 PL:ILLUMINA PU:HWUSI-EAS1707-615LHAAXX-L002 LB:80 DT:2010-05-05T20:00:00-0400 SM:SD37743 CN:UMCORE @PG ID:bwa VN:0.5.4 ALIGNMENT SECTION 8_96_444_1622 73 scaffold00005 155754 255 54M * 0 0 ATGTAAAGTATTTCCATGGTACACAGCTTGGTCGTAATGTGATTGCTGAGCCAG C@B5)5CBBCCBCCCBC@@7C>CBCCBCCC;57)8(@B@B>ABBCBC7BCC=> NM:i:0 8_80_1315_464 81 scaffold00005 155760 255 54M = 154948 0 AGTACCTCCCTGGTACACAGCTTGGTAAAAATGTGATTGCTGAGCCAGACCTTC B?@?BA=>@>>7;ABA?BB@BAA;@BBBBBBAABABBBCABAB?BABA?BBBAB NM:i:0 8_17_1222_1577 73 scaffold00005 155783 255 40M1116N10M * 0 0 GGTAAAAATGTGATTGCTGAGCCAGACCTTCATCATGCAGTGAGAGACGC BB@BA??>CCBA2AAABBBBBBB8A3@BABA;@A:>B=,;@B=A:BAAAA NM:i:0 XS:A:+ NS:i:0 8_43_1211_347 73 scaffold00005 155800 255 23M1116N27M * 0 0 TGAGCCAGACCTTCATCATGCAGTGAGAGACGCAAACATGCTGGTATTTG #>8<=<@6/:@9';@7A@@BAAA@BABBBABBB@=<A@BBBBBBBBCCBB NM:i:2 XS:A:+ NS:i:0 8_32_1091_284 161 scaffold00005 156946 255 54M = 157071 0 CGCAAACATGCTGGTAGCTGTGACACCACATCAACAGCTTGACTATGTTTGTAA BBBBB@AABACBCA8BBBBBABBBB@BBBBBBA@BBBBBBBBBA@:B@AA@=@@ NM:i:0 Quality Start position Sequence Read name

  35. Steps in resequencing analysis 1) Setup programs, data 2,3,4) map reads to a reference find best placement of reads bam file realign indels remove duplicates 5) recalibrate alignments recalibrate base quality bam file statistical algorithms 6) identify/call variants to detect true variants vcf file

  36. Steps in resequencing analysis 1) Setup programs, data 2,3,4) map reads to a reference find best placement of reads bam file realign indels remove duplicates 5) recalibrate alignments recalibrate base quality bam file statistical algorithms 6) identify/call variants to detect true variants vcf file

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