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==Export Mismatch from Chamber 15 to chamber 18 and CGI data== * remove SNV that are found in CGI and report mismatch to both alternative chamber and CGI $ perl /home/ericchu/softwares/removeduplicateline.pl PGP1_21_ch15.unique.HLA.mismatch18.vcf pgp1f_hg19_vcf_fixedCompleteGenomics_withHeaders_final.HLA.vcf PGP1_21_ch15.unique.HLA.mismatch18nCGI.vcf & $ wc -l PGP1_21_ch15.unique.HLA.mismatch18nCGI.vcf 499 PGP1_21_ch15.unique.HLA.mismatch18nCGI.vcf $ perl /home/ericchu/softwares/removeduplicateline.pl PGP1_21_ch18.unique.HLA.mismatch15.vcf pgp1f_hg19_vcf_fixedCompleteGenomics_withHeaders_final.HLA.vcf PGP1_21_ch18.unique.HLA.mismatch15nCGI.vcf & $ wc -l PGP1_21_ch18.unique.HLA.mismatch15nCGI.vcf 350 PGP1_21_ch18.unique.HLA.mismatch15nCGI.vcf * Extracting those mismatch SNV that were covered by the other chamber $ perl /home/ericchu/softwares/countVCFinbed2.pl PGP1_21_ch18.sorted.haplotype2.HLA.bed PGP1_21_ch15.unique.HLA.mismatch18nCGI.vcf PGP1_21_ch15vcfuniqueinch18bed.txt & $ awk '!seen[$0]++' PGP1_21_ch15vcfuniqueinch18bed.txt > PGP1_21_ch15vcfuniqueinch18bedall.txt $ wc -l PGP1_21_ch15vcfuniqueinch18bedall.txt 251 PGP1_21_ch15vcfuniqueinch18bedall.txt $ perl /home/ericchu/softwares/countVCFinbed2.pl PGP1_21_ch15.sorted.haplotype2.HLA.bed PGP1_21_ch18.unique.HLA.mismatch15nCGI.vcf PGP1_21_ch18vcfuniqueinch15bed.txt & $ awk '!seen[$0]++' PGP1_21_ch18vcfuniqueinch15bed.txt > PGP1_21_ch18vcfuniqueinch15bedall.txt $ wc -l PGP1_21_ch18vcfuniqueinch15bedall.txt 216 PGP1_21_ch18vcfuniqueinch15bedall.txt /home/kunzhang/softwares/samtools-0.1.19/samtools mpileup -BQ0 -d1000000 -f /GenomeDB/Homo_sapiens/UCSC/hg19/Sequence/WholeGenomeFasta/genome.fa PGP1_21_ch15.unique.fragment.bam -l PGP1_21_ch15vcfuniqueinch18bed.txt > PileupCh15HLAmismatchCh18.txt /home/kunzhang/softwares/samtools-0.1.19/samtools mpileup -BQ0 -d1000000 -f /GenomeDB/Homo_sapiens/UCSC/hg19/Sequence/WholeGenomeFasta/genome.fa PGP1_21_ch18.unique.fragment.bam -l PGP1_21_ch15vcfuniqueinch18bed.txt > PileupCh15HLAmismatchCh18complement.txt perl /home/ericchu/softwares/countbase.pl PileupCh15HLAmismatchCh18.txt AlleleCountCh15HLAmismatchCh18.txt perl /home/ericchu/softwares/countbase.pl PileupCh15HLAmismatchCh18complement.txt AlleleCountCh15HLAmismatchCh18complement.txt * Example first 20 lines in PileupCh15HLAmismatchCh18.txt, where these chamber 15 SNV were found called reference in chamber 18. chr6 30543198 A 1 ^]G B chr6 30543443 A 1 g I chr6 30552647 g 1 c$ B chr6 30555378 c 1 ^]G B chr6 30555477 t 1 G B chr6 30571649 A 4 .Gg, FFFI chr6 30590696 C 53 t$,.,t.T.,.,t.tT.,.ttTTT.T,,t,,t,..,...,,,.,.,,,.,,.., BBBFFBF0FFF<FFFFBBFF7FFFFFIIFFFFBBF<IBFFFIhFBBBFFBFFB chr6 30591764 c 45 .$,,,.,,.,T,,,..,.,t,,..,T..,,,,.T..tt,tt,t.T. FBIFFFIBIFIFF<IFFIIIFIIFBFIFBFFIBBFBB<BFFBFFB chr6 30611816 A 4 G.g. BFFI chr6 30611991 A 1 g B chr6 30612004 C 1 t B chr6 30612008 C 1 t F chr6 30612929 C 6 T.t.,. FFIFBF chr6 30614744 T 2 Cc FF chr6 30614938 G 1 A W chr6 30615658 A 4 T.,t FBBB chr6 30616279 a 1 G B chr6 30616288 t 1 G B chr6 30616292 c 1 T F chr6 30617172 G 1 a B * Example first 20 lines in PileupCh15HLAmismatchCh18complement.txt, where the reference was called in chamber 18. We can see that the confidence of these reference calls were much higher. Therefore, we only look at those AlleleCount in chamber 18 (AlleleCountCh15HLAmismatchCh18complement.txt) that were not high in confidence (where alternate ALT was called.) chr6 30543198 A 9 ..,,,,... 0BIIFFIFI chr6 30543443 A 14 ,.....,,...,., BFF<FIIBFBIFFF chr6 30552647 g 6 ..,,.^]. B<IFFB chr6 30555378 c 13 ,$..,,,,..,... BFFIIIFIIBFIF chr6 30555477 t 14 .,.,.,,.,,,,,^]. <FBFFIFFFFFBBB chr6 30571649 A 1 . F chr6 30590696 C 20 ..,,..,.,.,...,.,,^].^]. FFBBBFFIFIFFfIF0FBB< chr6 30591764 c 33 ..,..,,.,,,,,.,..,,,.,.,.,.,.,,,. FFFFFIIFIFFIIFFFFFIIIBFBFBI0IBFfB chr6 30611816 A 56 .$......,,,.....,,,,.,.C.,,,,...,,..,,,.,,,..,,..,..,,,,, FBBBFFBIII<BIFFFBFIIII'IIBIFIIFFFIIFBFIFFFIIFBIIBIFBFBFF chr6 30611991 A 35 ,,,...,,.,,,...,.....,,,,,....,..., BBFBFFIIFIFIFBIIFI7IIFFFF'IIIIB<FFf chr6 30612004 C 30 .,,.,,,...,.....,,,,,....,..., FFBFIFIBBFIFFBIIBFBF'IIIF<0BIf chr6 30612008 C 30 ,$,.,,,...,.....,,,,,....,...,, BFFFBIFFFIFF<FFIFFI<IIIFBFFIfB chr6 30612929 C 34 g,.....,..,....,.,..,,..........., BFBFFFIFIFIFIFIII0III<IFIIIIIIFIF7 chr6 30614744 T 10 ,..,,,,,,, <BFB<BFBF< chr6 30614938 G 2 ,. FI chr6 30615658 A 17 ,,...,.,,.....,,^], <FBF<FBFB<IBFF<B< chr6 30616279 a 26 .....,,.,,....,,,,.,,.,.,, 0FF<BIIFIIFIIIBBF0F<BF<BB' chr6 30616288 t 31 ....,,.,,....,,,,.,,.,.,,.,,^\.^],^], FFBFIFBIFFFIFB<FBFBBIBFB<F'0B0< chr6 30616292 c 31 .$..,,.,,....,,,,.,,.,.,,.,,.,,^]. F<<FFFIFFFIFFBFFIBFIBIB'F''FB0B chr6 30617172 G 36 .$.$.....,,,,.,..,,,,,,,,..,.,..,..,^].^], B0FBBBFIIIFFFFIIFIFIIFFII0FBIIB<FBB< chr6 30619551 a 181 ,,,,..,...,,,.,,,,,...,,,,.......,.,,,,..$.,,,,,,,.,,...,,,..,,..,,,,,,.....,...,,,..,.....,,,.,,,,,....,,...,,.,...,,,...,,,..,,,,..,,..,,...,,,,.,,,.,,,...g..,,,,,.,,g.,,,,,,,,..^], B0BF<FFBFBFFFBFFFFFFBBFF<FBBF<F0FIFIIII<F<IIIFIII<FF<FFIIIIIIIIIIBFIIBIFFIFIFIFIFF0FBIIIIIBFFIIIIFfIII_IFIIFIIIf<IIIIFBIFIFIIIIBIIIFIFFFIIIIIFFeFFFBFFFFFIIF<FFBBBBFFFF0FFFFFBBFBFBf chr6 30621413 g 66 aa...........,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,...,,.,,,,,...,,^].^], FFIIBFFIIIIFhIIIIBFIFIFIIBF<IFFIFIFIIBIIFFFIIIIFIIIFFFBFfBBFFFFfBB * These are the ones that are low confidence in Chamber 18 (AlleleCountCh15HLAmismatchCh18complement.txt). The criteria of confidence here was ALT base frequency > REF base. chr6 31106499 G A Depth = 1 A1, C0, G0, T0 chr6 31239752 C T Depth = 1 A0, C0, G0, T1 chr6 31240473 T C Depth = 3 A0, C2, G0, T1 chr6 31240489 A G Depth = 3 A1, C0, G2, T0 chr6 31240490 G C Depth = 3 A0, C2, G1, T0 chr6 31240496 G C Depth = 3 A0, C2, G1, T0 chr6 31240497 G A Depth = 3 A2, C0, G1, T0 chr6 31240498 T C Depth = 3 A0, C2, G0, T1 chr6 31240592 T C Depth = 4 A0, C3, G0, T1 chr6 31240593 G A Depth = 4 A3, C0, G1, T0 chr6 31240759 A G Depth = 4 A1, C0, G3, T0 chr6 31240789 G A Depth = 7 A6, C0, G1, T0 chr6 31240854 C A Depth = 3 A2, C1, G0, T0 chr6 31240864 C T Depth = 3 A0, C1, G0, T2 chr6 31241260 T C Depth = 2 A0, C2, G0, T0 chr6 31242174 G A Depth = 1 A1, C0, G0, T0 chr6 31323766 T C Depth = 1 A0, C1, G0, T0 chr6 31325526 G A Depth = 2 A2, C0, G0, T0 chr6 31325880 G A Depth = 2 A2, C0, G0, T0 chr6 31325881 A G Depth = 2 A0, C0, G2, T0 * The first SNV was dropped in chamber 18 and called a reference in concensus because of low (1) base quality. But we can actually see that both chambers were calling the same SNV. In PileupCh15HLAmismatchCh18.txt, chr6 31106499 G 11 AAAAaAAAaAa B'BFBIFIBB7 In PileupCh15HLAmismatchCh18complement.txt, chr6 31106499 G 1 a 7 * The second SNV was also dropped in chamber 18 for the same reason chr6 31239752 C 72 TttTTTtTTTttttTttTTTTTtttTTTTtttttTTTttttTtttttttttttttttTtT.TTTttTTtttT 7FFF7<B777FBFF'FI<7FB7BII7BFBIFIIFB7BFFFFFFFBBBBBBBBFBF<FF<IBIBIBBFFB'BB chr6 31239752 C 1 T 7 * The next 6 mismatch have a pattern. This is definitely a mapping issue! Two very similar reads were mapped to same position. chamber 15 chr6 31240473 T 54 ....,...,...,,,,..,,.C,,,.cc,cCccc,c.CCCCCCCCC,C,,CC.C <B<<BB0<IF7<FIBFBBFI<FIIIFFFIFIFFFFFIBIIFFFFFFdFFFF<<B chr6 31240489 A 56 .,...,,,,..,,.G,,,.gg,gGggg,g.GGGGGGGGG,G,,GG.Ggg,,...^].^], FBFFFBFFIIIFIIFIIFIIIFIFBIIIIIBFIFIFIFBfFFFIFIIFFFFFFFIF chr6 31240490 G 56 .$,...,,,,..,,.C,,,.tc,cCccc,c.CCCCCCCCC,C,,CC.Ccc,,...., FBFFF<F<FIIFFIFIIFIIIFFF<IIFII7IIFIFIF<dIFFFBIIFFFFFFBIF chr6 31240496 G 58 ...,,,,..,,.C,,,.cc,cCccc,c.CCCCCCCCC,C,,CC.Ccc,,....,,,,^\, FBFFIIIFIIIIIIIIIIFFFIIIFIII'IBFIIIIIfIFIIIIIFFBBII0IFFFFB chr6 31240497 G 58 ...,,,,..,,.A,,,.aa,aAaaa,a.AAAAAAAAA,A,,AA.Aaa,,....,,,,, FFFBIFIFIIIIFIIFFIFIFIIIFFII0IFFIIIFFdIIIIFIIFF<<IIBFFFFBB chr6 31240498 T 58 ...,,,,..,,.C,,,.cc,cCccc,c.CCCCCCCCCcC,,CC.Ccc,,....,,,,, B<BBFFI<<IIFIIIFFIIBIIIIIIIF'IFIIIIIIhIFIIFFIIFB'BF<FFFFBB chamber 18 chr6 31240473 T 3 .cc FBF chr6 31240489 A 3 .gg FFF chr6 31240490 G 3 .cc BBF chr6 31240496 G 3 .cc B<F chr6 31240497 G 3 .aa BBF chr6 31240498 T 3 .cc BFI * Next two. Again mapping issue. chamber 15 chr6 31240592 T 68 ,,,,.c$c$c$.C.CCc,,c,,CC.cccc,C,cccc,cc.c,.CC.C.,...,.,,..,..,,.,,...., BBBFFBBB<FF<FBIIIIIFFFFIF7III<FIIFIIFIIIBFIIFIIFBIIIIFIBFFBFIFBFBBBB chr6 31240593 G 66 ,$,,,..A.AAa,,a,,AA.aaaa,A,aaaa,aa.a,.$AA.A.,...,.,,..,..,,.,,....,^]. BBBBFBFFBFIIFFFIFFFFIIFIIIIFFIBIIFIIIFFIIFIIIFIIIIIIBFFFFIFFFFBBFB chamber 18 chr6 31240592 T 4 ccc, IFIF chr6 31240593 G 4 aaa, IFIF * Then now, the others with higher confidence reference call in Chamber 18 may also have the same mapping issue. /home/kunzhang/softwares/samtools-0.1.19/samtools mpileup -BQ0 -d1000000 -f /GenomeDB/Homo_sapiens/UCSC/hg19/Sequence/WholeGenomeFasta/genome.fa PGP1_21_ch18.unique.fragment.bam -l PGP1_21_ch18vcfuniqueinch15bed.txt > PileupCh18HLAmismatchCh15.txt & /home/kunzhang/softwares/samtools-0.1.19/samtools mpileup -BQ0 -d1000000 -f /GenomeDB/Homo_sapiens/UCSC/hg19/Sequence/WholeGenomeFasta/genome.fa PGP1_21_ch15.unique.fragment.bam -l PGP1_21_ch18vcfuniqueinch15bed.txt > PileupCh18HLAmismatchCh15complement.txt perl /home/ericchu/softwares/countbase.pl PileupCh18HLAmismatchCh15.txt AlleleCountCh18HLAmismatchCh15.txt perl /home/ericchu/softwares/countbase.pl PileupCh18HLAmismatchCh15complement.txt AlleleCountCh18HLAmismatchCh15complement.txt
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