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Hi all,
I am consistently seeing the following pattern that alerts me to a systematic problem in some step in my sequencing/filtering pipeline. I used the Illumina MiSeq to deep sequence a mixed bacterial population, and did this in two ways -- I prepared three Nextera XT libraries on the same day (technical replicates) and also six months apart from each other (different-day-replicates). I am aligning to a reference genome using bwa, and then creating an mpileup file using samtools mpileup -f REF.fna > output.mpileup. When call SNPs (I am requiring > 12X coverage and > 90% of the reads must be the same variant base) from the technical replicates, about 99% of positions agree across all three technical replicate libraries (around 400 sites). When I compare the different-day-replicates (same tube of genomic DNA prepared 6 months apart), there are around 300 sites that are discordant. I am now trying to figure out if there is a systematic difference between the discordant and concordant SNPs.
Many of the SNPs, in both the discordant and concordant categories, follow this pattern:
Here's a library where position 3448348 is a SNP:
gi|448814763|ref|NC_000962.3| 3448343 C 25 .,,,,,,...,,,,.,,,,.,,... HCCGEGGHFGGECGHGGGGHGGGFA
gi|448814763|ref|NC_000962.3| 3448344 G 25 .,,,,,,...,,,,.,,,,.,,... GFFGFGGGFGHHFHGHHHHGHHFCB
gi|448814763|ref|NC_000962.3| 3448345 G 26 .,,,,,,...,,,,.,,,,.,,...^], GCCGGCGGDGGGGGGGGGGGDGEBA8
gi|448814763|ref|NC_000962.3| 3448346 C 26 .,,,,,,...,,,,.,,,,.,,..., GBBGGEGG@CGEGGGGGGGG/GAGB9
gi|448814763|ref|NC_000962.3| 3448347 A 26 .$,,,,,,...,,,,.,,,,.,,..., E>==9999<99F9999999999?9>6
gi|448814763|ref|NC_000962.3| 3448348 G 25 aaaaaaAAAaaaaAaaaaAaaAAAa >==9999=9919999999999?9>6
gi|448814763|ref|NC_000962.3| 3448349 C 25 ,,,,,,...,,,,.,,,,.,,..., CCCAGGHHCGEGGHGGGGHGGFGB<
gi|448814763|ref|NC_000962.3| 3448350 G 26 ,,,,,,...,,,,.,,,,.,,...,^]. CCCGFGGG@GEGGGGGGGGGGFGBGA
gi|448814763|ref|NC_000962.3| 3448351 G 26 ,,,,,,...,,,,.,,,,.,,...,. BCBGGGGCGGEGGGGGGCGCG?GBGB
gi|448814763|ref|NC_000962.3| 3448352 G 26 ,$,,,,,...,,,,.,,,,.,,...,. @CCGEGGGGGGFGGGGGFGCGEGBGC
gi|448814763|ref|NC_000962.3| 3448353 T 26 ,,,,,...,,,,.,,,,.,,...,.^>. CCB>GGDGGEGGGGGGGG@GGGGGA7
And a library where it's not a SNP:
gi|448814763|ref|NC_000962.3| 3448343 C 26 ..,..,...,,,.,.,..,,.,.,,, HHDGHGHHHGCGHGH/HH</HGGGDG
gi|448814763|ref|NC_000962.3| 3448344 G 26 ..,..,...,,,.,.,..,,.,.,,, GGCGGGGAGHHHGHG0GF0FGHFGFH
gi|448814763|ref|NC_000962.3| 3448345 G 26 ..,..,...,,,.,.,..,,.,.,,, GGAGGGFAGGGGGGG?GG/GGGGGDG
gi|448814763|ref|NC_000962.3| 3448346 C 26 ..,..,...,,,.,.,..,,.,.,,, GG@GGGCCGGGEGGG/GG/EGGGGFG
gi|448814763|ref|NC_000962.3| 3448347 A 26 ..,..,...,,,.,.,..,,.,.,,, GCDGGGCGGGG?GGG/GG<CGGGGFG
gi|448814763|ref|NC_000962.3| 3448348 G 26 .$.,..,...,,,.,.,..c,.,.,,, CFAGGCF@GGGEGGG/GG/GGGGGGG
gi|448814763|ref|NC_000962.3| 3448349 C 25 .,..,...,,,.,.,..,,.,.,,, GAGGCGFGGFGGGG/GGEEGGGGGG
gi|448814763|ref|NC_000962.3| 3448350 G 25 .,..,...,,,.,.,..,,.,.,,, G>GGCGGGGEGGGG>GG>EGGGGGG
gi|448814763|ref|NC_000962.3| 3448351 G 25 .,..,...,,,.,.,..,,.,.,,, G3GGCGGGG0GGGG>GG>>GHGGGG
gi|448814763|ref|NC_000962.3| 3448352 G 26 .,..,...,,,.,.,..,,.,.,,,^], G3FGCGGGGGGGGG/GG//GHGGCGE
gi|448814763|ref|NC_000962.3| 3448353 T 26 .$,..,...,,,.,.c..,,.,.,,,, :3GGCGGGGGEGGG/GG//GHGG@GG
gi|448814763|ref|NC_000962.3| 3448354 C 26 ,$..,...,,,.,.,..,,.,.,,,,^], >EECGGGGGCGGG/GG//G2GGCGGB
gi|448814763|ref|NC_000962.3| 3448355 C 25 .$.$,...,,,.,.,..,,.a.,,,,, AACHHAGGGHGH/HH//H-HGCGGE
gi|448
Example 2: Position 67792:
gi|448814763|ref|NC_000962.3| 67788 A 19 ..,,,,,,,..,,.,..,. GGFFGFGGGGCHHCGCBGC
gi|448814763|ref|NC_000962.3| 67789 T 19 ..,,,,,,,..,,.,..,. GGCDGGGGGGGGGFGCCGC
gi|448814763|ref|NC_000962.3| 67790 T 19 ..,,,,,,,..,,.,..,. GCCBGGGGGGGGGGGCCGD
gi|448814763|ref|NC_000962.3| 67791 G 20 .$.,,,,,,,..,,.,..,.^]. EHCBGGGGGHHGGHGFFGF1
gi|448814763|ref|NC_000962.3| 67792 C 19 TtttttttTTttTtTTtTT 8=>9:9998899999:9:0
gi|448814763|ref|NC_000962.3| 67793 C 19 .,,,,,,,..,,.,..,.. 8=>9:9998899999:9:;
gi|448814763|ref|NC_000962.3| 67794 G 19 .,,,,,,,..,,.,..,.. GFFGGGGGGGGHAHGGHDA
library where it's not a SNP:
gi|448814763|ref|NC_000962.3| 67788 A 47 ,$,$.....,,.,,,,,,....,.,,.....,,,,........,,.,.. B1GGGGGFG@GGGG1GGGC/HGH1EGGGGFFHHGGGGGGDDGGBGCA
gi|448814763|ref|NC_000962.3| 67789 T 45 .....,,.,,,,,,....,.,,.....,,,,........,,.,.. GGGGGDBDGGCGG?GGG/EGE/GGGGEGCGGGGGGGGCAGGBGCB
gi|448814763|ref|NC_000962.3| 67790 T 45 .....,,.,,,,,,....,.,,.....,,,,........,,.,.. GGGGGCBFGGGEGGGGG/?GE/GGGGGG@GGGGGGGGC?GGBGCB
gi|448814763|ref|NC_000962.3| 67791 G 45 .....,,.,,,,,,....,.,,.....,,,,........,,.,.. HHGHHCBHGGGGEGHHH1?FE/HGHHBGGGGHFHHHGFFF@FGFF
gi|448814763|ref|NC_000962.3| 67792 C 46 .....,,.,,,,,,....,.,,.....,,,,........,,.,..^], HHHHHBBHGGGGEGHHG1EH@/GHHHGGGGCHBHHHHGDGCFGFF@
gi|448814763|ref|NC_000962.3| 67793 C 46 .....,,.,,,,,,....,.,,.....,,,,........,,.,.., HHHHHBBHGFFGEGHHH1EHCEFHHHGGGEGHEHHHHGGGCFGFF-
gi|448814763|ref|NC_000962.3| 67794 G 46 .....,,.,,,,,,...C,.,,.....,,,,........,,.,.., GGGGGFFGGGGGBGGGC1HGFGECGGGHHGGG/GGGGGEHHBECBF
gi|448814763|ref|NC_000962.3| 67795 C 46 .$....,,.,,,,,,....,.,,.....,,,,........,,.,.., EGGGGCFGFFGFGFGGG<HGHAGCGGGHHFHGEGGGGGEFGBHCBB
Two things are weird. In the first library in each example, the variant position has a local drop in Phred quality score compared to the rest of the positions, AND the position just above it or just below it seems to have the exact same (lower) Phred scores. But this position is not a SNP in the second library, and doesn't have the same drop in Phred sores.
A contrasting example -- this position is discordant between the different runs, but doesn't show the same drop in Phred scores. Here's a library where it's a SNP:
gi|448814763|ref|NC_000962.3| 23611 G 41 ,,......,,,,,,.,,,,,*,,.,.,,..,,,.,..,,,. CAGGGGGGGGEGGGGGGGGF!GGGGEGGGGGGGGGACGCD'
gi|448814763|ref|NC_000962.3| 23612 G 41 ,,......,,,,,,.,,,,,*,,.,.,,..,,,.,..,,,. BAGGC/GGGFAGGGGGGGGA!GGGGEFEGGGGGGG?CDGD;
gi|448814763|ref|NC_000962.3| 23613 C 41 ,,......,,,,,,.,,,,,*,,.,.,,..,,,.,..,,,. BAGGC<GGGFBGGEGGGGF/!GGGGGE?GGGEEGGAC/D<G
gi|448814763|ref|NC_000962.3| 23614 G 41 ,$,......,,,,,,.,,,,,*,,.,.,,..,,,.,..,,,. CFGGF<GGHHHGHGGHHHHF!HHGHCHGGFGGHGHECGG1G
gi|448814763|ref|NC_000962.3| 23615 C 40 ,......,,,,,,.,,,,,*,,.,.,,..,,,.,..,,,. FGGGCCGHGHHHDGHHFHF!GGGHFHFGGHBHGHECBF1G
gi|448814763|ref|NC_000962.3| 23616 A 40 ,......,,,,,,.,,,,,*,,.,.,,..,,,.,..,,,. FGGFCGGHHHHHHGHHHHF!HHGHEH2GEFHHGH0GFFGG
gi|448814763|ref|NC_000962.3| 23617 T 40 ,......,,,,,,.,,,,,*,,.,.,,..,,,.,..,,,. BGG??DGHHHHHGGFHHHG!FHGHGHDGEHHHGH0CGGFG
gi|448814763|ref|NC_000962.3| 23618 C 40 gGGGGGGggggggGggggg*ggGgGggGGgggGgGGgggG BGGGCGGHHHHHHGHHHHF!HHGHGHFGEHGHGHEGHHGG
gi|448814763|ref|NC_000962.3| 23619 A 40 ,......,,,,,,.,,,,,*,,.,.,,..,,,.,..,,,. BHHHHHHHHHHHHFHHHH@!HHHHGH2HHGFHHH1GGHGH
gi|448814763|ref|NC_000962.3| 23620 T 40 ,......,,,,,,.,,,,,*,,.,.,,..,,,.,..,,,. BGHGDFHGAGGGGHGGGG/!GGHGHG/HHGDGHGAGEGDH
and here's a library where it's not:
gi|448814763|ref|NC_000962.3| 23610 C 25 ,$,.......,,,.,,.,,,,,.,,, AGGGGGGCGGGGGGGGGG/GGCFDG
gi|448814763|ref|NC_000962.3| 23611 G 24 ,.......,,,A,,.,,,,a.,,, GGGGGGCGGGG9GGGGE/G9GGBG
gi|448814763|ref|NC_000962.3| 23612 G 24 ,.......,,,.,,.,,,,,.,,, BGGGGG/GGGG9GGGGF/G9GDGG
gi|448814763|ref|NC_000962.3| 23613 C 24 ,.......,,,.,,.,,,,,.,,, BGGGGGBGGGGGGGGG@CGFG/CG
gi|448814763|ref|NC_000962.3| 23614 G 24 ,.......,,,.,,.,,,,,.,,, FGGGGGCFHHHGFHGHGHHHGFDH
gi|448814763|ref|NC_000962.3| 23615 C 25 ,.......,,,.,,.,,,,,.,,,^], BGGGGGGGHHHGHHGFFFHHG4FHE
gi|448814763|ref|NC_000962.3| 23616 A 25 ,.......,,,.,,.,,,,,.,,,, FGGGGGCGHHHGHHGHHHHHGHFHG
gi|448814763|ref|NC_000962.3| 23617 T 25 ,.......,,,.,,.,,,,,.,,,, FGGGGG?GHHHGHHGGHHHHGHHHB
gi|448814763|ref|NC_000962.3| 23618 C 25 ,.......,,,.,,.,,,,,.,,,, FGGGGG@GGHHGHHGGHGHHGGFHH
gi|448814763|ref|NC_000962.3| 23619 A 25 ,.......,,,.,,.,,,,,.,,,, BHGHHHFHHHHHHHHHHGHHGHHHH
gi|448814763|ref|NC_000962.3| 23620 T 25 ,.......,,,.,,.,,,,,.,,,, BHHHHHFHGGGFGGHGGEGGGFDGG
gi|448814763|ref|NC_000962.3| 23621 G 25 ,.......,,,.,,.,,,,,.,,,, BHHHHHHHGGGHGGHGG?GGHG@GG
gi|448814763|ref|NC_000962.3| 23622 A 25 ,.......,,,.,,.,,,,,.,,,, BHHHHHHHGGGHGGHGGEGGHGGGG
gi|448814763|ref|NC_000962.3| 23623 C 25 ,$.......,,,.,,.,,,,,.,,,, >HHHHHHHGGGHGGHGGFGGHGGGG
gi|448814763|ref|NC_000962.3| 23624 C 24 .......,,,.,,.,,,,,.,,,, HHHHHHHGGGHGGHGGGGGHGGGG
gi|448814763|ref|NC_000962.3| 23625 G 24 .......,,,.,,.,,,,,.,,,, GGGGGGGGGGGGGGGGGGGGGGGG
I am seeing this "local drop" in many of the discordant SNPs. Any idea why this might be happening? And why it might be bleeding over to neighboring positions? Is this cause for alarm that these positions might be false positives?Any other suggestions for sorting out these discordant positions?
Thanks! Any thoughts are much appreciated!
I am consistently seeing the following pattern that alerts me to a systematic problem in some step in my sequencing/filtering pipeline. I used the Illumina MiSeq to deep sequence a mixed bacterial population, and did this in two ways -- I prepared three Nextera XT libraries on the same day (technical replicates) and also six months apart from each other (different-day-replicates). I am aligning to a reference genome using bwa, and then creating an mpileup file using samtools mpileup -f REF.fna > output.mpileup. When call SNPs (I am requiring > 12X coverage and > 90% of the reads must be the same variant base) from the technical replicates, about 99% of positions agree across all three technical replicate libraries (around 400 sites). When I compare the different-day-replicates (same tube of genomic DNA prepared 6 months apart), there are around 300 sites that are discordant. I am now trying to figure out if there is a systematic difference between the discordant and concordant SNPs.
Many of the SNPs, in both the discordant and concordant categories, follow this pattern:
Here's a library where position 3448348 is a SNP:
gi|448814763|ref|NC_000962.3| 3448343 C 25 .,,,,,,...,,,,.,,,,.,,... HCCGEGGHFGGECGHGGGGHGGGFA
gi|448814763|ref|NC_000962.3| 3448344 G 25 .,,,,,,...,,,,.,,,,.,,... GFFGFGGGFGHHFHGHHHHGHHFCB
gi|448814763|ref|NC_000962.3| 3448345 G 26 .,,,,,,...,,,,.,,,,.,,...^], GCCGGCGGDGGGGGGGGGGGDGEBA8
gi|448814763|ref|NC_000962.3| 3448346 C 26 .,,,,,,...,,,,.,,,,.,,..., GBBGGEGG@CGEGGGGGGGG/GAGB9
gi|448814763|ref|NC_000962.3| 3448347 A 26 .$,,,,,,...,,,,.,,,,.,,..., E>==9999<99F9999999999?9>6
gi|448814763|ref|NC_000962.3| 3448348 G 25 aaaaaaAAAaaaaAaaaaAaaAAAa >==9999=9919999999999?9>6
gi|448814763|ref|NC_000962.3| 3448349 C 25 ,,,,,,...,,,,.,,,,.,,..., CCCAGGHHCGEGGHGGGGHGGFGB<
gi|448814763|ref|NC_000962.3| 3448350 G 26 ,,,,,,...,,,,.,,,,.,,...,^]. CCCGFGGG@GEGGGGGGGGGGFGBGA
gi|448814763|ref|NC_000962.3| 3448351 G 26 ,,,,,,...,,,,.,,,,.,,...,. BCBGGGGCGGEGGGGGGCGCG?GBGB
gi|448814763|ref|NC_000962.3| 3448352 G 26 ,$,,,,,...,,,,.,,,,.,,...,. @CCGEGGGGGGFGGGGGFGCGEGBGC
gi|448814763|ref|NC_000962.3| 3448353 T 26 ,,,,,...,,,,.,,,,.,,...,.^>. CCB>GGDGGEGGGGGGGG@GGGGGA7
And a library where it's not a SNP:
gi|448814763|ref|NC_000962.3| 3448343 C 26 ..,..,...,,,.,.,..,,.,.,,, HHDGHGHHHGCGHGH/HH</HGGGDG
gi|448814763|ref|NC_000962.3| 3448344 G 26 ..,..,...,,,.,.,..,,.,.,,, GGCGGGGAGHHHGHG0GF0FGHFGFH
gi|448814763|ref|NC_000962.3| 3448345 G 26 ..,..,...,,,.,.,..,,.,.,,, GGAGGGFAGGGGGGG?GG/GGGGGDG
gi|448814763|ref|NC_000962.3| 3448346 C 26 ..,..,...,,,.,.,..,,.,.,,, GG@GGGCCGGGEGGG/GG/EGGGGFG
gi|448814763|ref|NC_000962.3| 3448347 A 26 ..,..,...,,,.,.,..,,.,.,,, GCDGGGCGGGG?GGG/GG<CGGGGFG
gi|448814763|ref|NC_000962.3| 3448348 G 26 .$.,..,...,,,.,.,..c,.,.,,, CFAGGCF@GGGEGGG/GG/GGGGGGG
gi|448814763|ref|NC_000962.3| 3448349 C 25 .,..,...,,,.,.,..,,.,.,,, GAGGCGFGGFGGGG/GGEEGGGGGG
gi|448814763|ref|NC_000962.3| 3448350 G 25 .,..,...,,,.,.,..,,.,.,,, G>GGCGGGGEGGGG>GG>EGGGGGG
gi|448814763|ref|NC_000962.3| 3448351 G 25 .,..,...,,,.,.,..,,.,.,,, G3GGCGGGG0GGGG>GG>>GHGGGG
gi|448814763|ref|NC_000962.3| 3448352 G 26 .,..,...,,,.,.,..,,.,.,,,^], G3FGCGGGGGGGGG/GG//GHGGCGE
gi|448814763|ref|NC_000962.3| 3448353 T 26 .$,..,...,,,.,.c..,,.,.,,,, :3GGCGGGGGEGGG/GG//GHGG@GG
gi|448814763|ref|NC_000962.3| 3448354 C 26 ,$..,...,,,.,.,..,,.,.,,,,^], >EECGGGGGCGGG/GG//G2GGCGGB
gi|448814763|ref|NC_000962.3| 3448355 C 25 .$.$,...,,,.,.,..,,.a.,,,,, AACHHAGGGHGH/HH//H-HGCGGE
gi|448
Example 2: Position 67792:
gi|448814763|ref|NC_000962.3| 67788 A 19 ..,,,,,,,..,,.,..,. GGFFGFGGGGCHHCGCBGC
gi|448814763|ref|NC_000962.3| 67789 T 19 ..,,,,,,,..,,.,..,. GGCDGGGGGGGGGFGCCGC
gi|448814763|ref|NC_000962.3| 67790 T 19 ..,,,,,,,..,,.,..,. GCCBGGGGGGGGGGGCCGD
gi|448814763|ref|NC_000962.3| 67791 G 20 .$.,,,,,,,..,,.,..,.^]. EHCBGGGGGHHGGHGFFGF1
gi|448814763|ref|NC_000962.3| 67792 C 19 TtttttttTTttTtTTtTT 8=>9:9998899999:9:0
gi|448814763|ref|NC_000962.3| 67793 C 19 .,,,,,,,..,,.,..,.. 8=>9:9998899999:9:;
gi|448814763|ref|NC_000962.3| 67794 G 19 .,,,,,,,..,,.,..,.. GFFGGGGGGGGHAHGGHDA
library where it's not a SNP:
gi|448814763|ref|NC_000962.3| 67788 A 47 ,$,$.....,,.,,,,,,....,.,,.....,,,,........,,.,.. B1GGGGGFG@GGGG1GGGC/HGH1EGGGGFFHHGGGGGGDDGGBGCA
gi|448814763|ref|NC_000962.3| 67789 T 45 .....,,.,,,,,,....,.,,.....,,,,........,,.,.. GGGGGDBDGGCGG?GGG/EGE/GGGGEGCGGGGGGGGCAGGBGCB
gi|448814763|ref|NC_000962.3| 67790 T 45 .....,,.,,,,,,....,.,,.....,,,,........,,.,.. GGGGGCBFGGGEGGGGG/?GE/GGGGGG@GGGGGGGGC?GGBGCB
gi|448814763|ref|NC_000962.3| 67791 G 45 .....,,.,,,,,,....,.,,.....,,,,........,,.,.. HHGHHCBHGGGGEGHHH1?FE/HGHHBGGGGHFHHHGFFF@FGFF
gi|448814763|ref|NC_000962.3| 67792 C 46 .....,,.,,,,,,....,.,,.....,,,,........,,.,..^], HHHHHBBHGGGGEGHHG1EH@/GHHHGGGGCHBHHHHGDGCFGFF@
gi|448814763|ref|NC_000962.3| 67793 C 46 .....,,.,,,,,,....,.,,.....,,,,........,,.,.., HHHHHBBHGFFGEGHHH1EHCEFHHHGGGEGHEHHHHGGGCFGFF-
gi|448814763|ref|NC_000962.3| 67794 G 46 .....,,.,,,,,,...C,.,,.....,,,,........,,.,.., GGGGGFFGGGGGBGGGC1HGFGECGGGHHGGG/GGGGGEHHBECBF
gi|448814763|ref|NC_000962.3| 67795 C 46 .$....,,.,,,,,,....,.,,.....,,,,........,,.,.., EGGGGCFGFFGFGFGGG<HGHAGCGGGHHFHGEGGGGGEFGBHCBB
Two things are weird. In the first library in each example, the variant position has a local drop in Phred quality score compared to the rest of the positions, AND the position just above it or just below it seems to have the exact same (lower) Phred scores. But this position is not a SNP in the second library, and doesn't have the same drop in Phred sores.
A contrasting example -- this position is discordant between the different runs, but doesn't show the same drop in Phred scores. Here's a library where it's a SNP:
gi|448814763|ref|NC_000962.3| 23611 G 41 ,,......,,,,,,.,,,,,*,,.,.,,..,,,.,..,,,. CAGGGGGGGGEGGGGGGGGF!GGGGEGGGGGGGGGACGCD'
gi|448814763|ref|NC_000962.3| 23612 G 41 ,,......,,,,,,.,,,,,*,,.,.,,..,,,.,..,,,. BAGGC/GGGFAGGGGGGGGA!GGGGEFEGGGGGGG?CDGD;
gi|448814763|ref|NC_000962.3| 23613 C 41 ,,......,,,,,,.,,,,,*,,.,.,,..,,,.,..,,,. BAGGC<GGGFBGGEGGGGF/!GGGGGE?GGGEEGGAC/D<G
gi|448814763|ref|NC_000962.3| 23614 G 41 ,$,......,,,,,,.,,,,,*,,.,.,,..,,,.,..,,,. CFGGF<GGHHHGHGGHHHHF!HHGHCHGGFGGHGHECGG1G
gi|448814763|ref|NC_000962.3| 23615 C 40 ,......,,,,,,.,,,,,*,,.,.,,..,,,.,..,,,. FGGGCCGHGHHHDGHHFHF!GGGHFHFGGHBHGHECBF1G
gi|448814763|ref|NC_000962.3| 23616 A 40 ,......,,,,,,.,,,,,*,,.,.,,..,,,.,..,,,. FGGFCGGHHHHHHGHHHHF!HHGHEH2GEFHHGH0GFFGG
gi|448814763|ref|NC_000962.3| 23617 T 40 ,......,,,,,,.,,,,,*,,.,.,,..,,,.,..,,,. BGG??DGHHHHHGGFHHHG!FHGHGHDGEHHHGH0CGGFG
gi|448814763|ref|NC_000962.3| 23618 C 40 gGGGGGGggggggGggggg*ggGgGggGGgggGgGGgggG BGGGCGGHHHHHHGHHHHF!HHGHGHFGEHGHGHEGHHGG
gi|448814763|ref|NC_000962.3| 23619 A 40 ,......,,,,,,.,,,,,*,,.,.,,..,,,.,..,,,. BHHHHHHHHHHHHFHHHH@!HHHHGH2HHGFHHH1GGHGH
gi|448814763|ref|NC_000962.3| 23620 T 40 ,......,,,,,,.,,,,,*,,.,.,,..,,,.,..,,,. BGHGDFHGAGGGGHGGGG/!GGHGHG/HHGDGHGAGEGDH
and here's a library where it's not:
gi|448814763|ref|NC_000962.3| 23610 C 25 ,$,.......,,,.,,.,,,,,.,,, AGGGGGGCGGGGGGGGGG/GGCFDG
gi|448814763|ref|NC_000962.3| 23611 G 24 ,.......,,,A,,.,,,,a.,,, GGGGGGCGGGG9GGGGE/G9GGBG
gi|448814763|ref|NC_000962.3| 23612 G 24 ,.......,,,.,,.,,,,,.,,, BGGGGG/GGGG9GGGGF/G9GDGG
gi|448814763|ref|NC_000962.3| 23613 C 24 ,.......,,,.,,.,,,,,.,,, BGGGGGBGGGGGGGGG@CGFG/CG
gi|448814763|ref|NC_000962.3| 23614 G 24 ,.......,,,.,,.,,,,,.,,, FGGGGGCFHHHGFHGHGHHHGFDH
gi|448814763|ref|NC_000962.3| 23615 C 25 ,.......,,,.,,.,,,,,.,,,^], BGGGGGGGHHHGHHGFFFHHG4FHE
gi|448814763|ref|NC_000962.3| 23616 A 25 ,.......,,,.,,.,,,,,.,,,, FGGGGGCGHHHGHHGHHHHHGHFHG
gi|448814763|ref|NC_000962.3| 23617 T 25 ,.......,,,.,,.,,,,,.,,,, FGGGGG?GHHHGHHGGHHHHGHHHB
gi|448814763|ref|NC_000962.3| 23618 C 25 ,.......,,,.,,.,,,,,.,,,, FGGGGG@GGHHGHHGGHGHHGGFHH
gi|448814763|ref|NC_000962.3| 23619 A 25 ,.......,,,.,,.,,,,,.,,,, BHGHHHFHHHHHHHHHHGHHGHHHH
gi|448814763|ref|NC_000962.3| 23620 T 25 ,.......,,,.,,.,,,,,.,,,, BHHHHHFHGGGFGGHGGEGGGFDGG
gi|448814763|ref|NC_000962.3| 23621 G 25 ,.......,,,.,,.,,,,,.,,,, BHHHHHHHGGGHGGHGG?GGHG@GG
gi|448814763|ref|NC_000962.3| 23622 A 25 ,.......,,,.,,.,,,,,.,,,, BHHHHHHHGGGHGGHGGEGGHGGGG
gi|448814763|ref|NC_000962.3| 23623 C 25 ,$.......,,,.,,.,,,,,.,,,, >HHHHHHHGGGHGGHGGFGGHGGGG
gi|448814763|ref|NC_000962.3| 23624 C 24 .......,,,.,,.,,,,,.,,,, HHHHHHHGGGHGGHGGGGGHGGGG
gi|448814763|ref|NC_000962.3| 23625 G 24 .......,,,.,,.,,,,,.,,,, GGGGGGGGGGGGGGGGGGGGGGGG
I am seeing this "local drop" in many of the discordant SNPs. Any idea why this might be happening? And why it might be bleeding over to neighboring positions? Is this cause for alarm that these positions might be false positives?Any other suggestions for sorting out these discordant positions?
Thanks! Any thoughts are much appreciated!
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