im newbie to NGS and linux. Im using sam file output from MapSplice in Cufflinks and facing the following problem :
**> Processing Locus chr9:43969287-43974380 [*********************** ] 95%SAM error on line 88768364: found spliced alignment without XS attribute
SAM error on line 88768374: found spliced alignment without XS attribute
SAM error on line 88768381: found spliced alignment without XS attribute
SAM error on line 88768383: found spliced alignment without XS attribute
SAM error on line 88768384: found spliced alignment without XS attribute
SAM error on line 88768389: found spliced alignment without XS attribute**
while the sam output file looks like this where some reads have XS attribute while others dont have:
TTGGCTAATCGTCAACCTGGAAGAGCTTTCCTCT @CCFDFFFGHHHFIIIIIJIJIIJGGIIJJCGHIJJJHIJJJC)*9BGIJIJIIIGIIIGFGIJIJJIJJJJHGHHFFDFECDDBCCBDDDDCDDDDD NM:i:0 IH:i:1 HI:i:1 XS:A:+ XF:Z:GTAG,
SRR959590.257 99 chr8 78240758 113 80M9S = 78240861 189 TACTGATGATGCCACTTGCCTGGCATGAAGAGGAGATGGTGGAGGTGGAGGAGGTGCAGATTTAGATGCTGAATTTACCTCAAAAGTGG CCCFFFFFGHHHHJJJJJJJJJJJJJJJJJGIJEHDHII?FHDHI0BG?FHGHI;FHIJJJJJIJHHHHHHFFFFDFFDE(.;CD>-;@ NM:i:1 IH:i:1 HI:i:1
SRR959590.257 147 chr8 78240861 118 86M = 78240758 -189 GGCGGGGGTGGTGGGGTTACAGGAGATCCAGCTCTAATAGGTGCAGGAGAAGGGGGAGGTGGAGGTATAGGTGTGGCAGGAGTTGT 0''DDDDDDDCBFHHGHHHEHHGHEFCHDCGC=)FCGDAF?0?0>IHFGEBJJJJIGJJJIJJJJIJJJJJJJHHHHHFFFFFCCC NM:i:0 IH:i:1 HI:i:1
SRR959590.377 83 chr13 49046592 139 82M110N19M = 49046362 -441 ATTTAGCATTTACTGTCAAATATCATAGGGATCCGCAAACTGACTCTGCTAGAATTGTGGGTTTTGAGGTTAAACCATACAGTATTAAACATGAATATGAG CDDDDDEFDCDCDCDEFFFFDEEFFDFFHHHJIJJIHGDJIHFJJJJJJIJIJJJIJIJJIIJJJJJIJJJGJHJJJGHJJJJJJJJJHHHHHFFFFFCCC NM:i:0 IH:i:1 HI:i:1 XS:A:+ XF:Z:GTAG,
SRR959590.377 163 chr13 49046362 135 98M = 49046592 441 TAACCTTCCCCTTGTTGTCCCCATTAGAAGATTAGATCAGGATTCTTCTACTGTTTATCAGCTTGGCTACCATGTTGGGCTCAAAGGGCAATATAGTG CCCFFFFFHHHGHIJIJJIJJJIIJGIIGIIIGIJJGIIIJI3B90?BGEBFDGGIGIGEIIIJEIIJJJIIIIIIGFEHFFFD).>7;?BDCDAABC NM:i:0 IH:i:1 HI:i:1
SRR959590.491 99 chr10 36684807 121 85M2I14M = 36684912 155 TGATTCATTATTTTTAGCGTCTCTAGTTCAAAACCGAACATGAAATTTTGGTTTCATTCGGCTCCTTTATGGAAGATGGATAAAATAAAAAAAAAAGAAAT @@@FDFFFDHGDHICEHIIGDGGHDEHGGGEHGEEG>@D@DFGGGGIIHIIFIGGGHIIID<DA?EHFBBEFC>CACDEDDDDDADCDDDDDDDDD&8??: NM:i:2 IH:i:4 HI:i:1
SRR959590.491 355 chr11 34180669 121 85M2I14M = 34180774 155 TGATTCATTATTTTTAGCGTCTCTAGTTCAAAACCGAACATGAAATTTTGGTTTCATTCGGCTCCTTTATGGAAGATGGATAAAATAAAAAAAAAAGAAAT @@@FDFFFDHGDHICEHIIGDGGHDEHGGGEHGEEG>@D@DFGGGGIIHIIFIGGGHIIID<DA?EHFBBEFC>CACDEDDDDDADCDDDDDDDDD&8??: NM:i:2 IH:i:4 HI:i:2
SRR959590.491 355 chr8 46854583 121 85M2I14M = 46854688 155 TGATTCATTATTTTTAGCGTCTCTAGTTCAAAACCGAACATGAAATTTTGGTTTCATTCGGCTCCTTTATGGAAGATGGATAAAATAAAAAAAAAAGAAAT @@@FDFFFDHGDHICEHIIGDGGHDEHGGGEHGEEG>@D@DFGGGGIIHIIFIGGGHIIID<DA?EHFBBEFC>CACDEDDDDDADCDDDDDDDDD&8??: NM:i:2 IH:i:4 HI:i:3
SRR959590.491 355 chr8 85697578 121 85M2I14M = 85697683 155 TGATTCATTATTTTTAGCGTCTCTAGTTCAAAACCGAACATGAAATTTTGGTTTCATTCGGCTCCTTTATGGAAGATGGATAAAATAAAAAAAAAAGAAAT @@@FDFFFDHGDHICEHIIGDGGHDEHGGGEHGEEG>@D@DFGGGGIIHIIFIGGGHIIID<DA?EHFBBEFC>CACDEDDDDDADCDDDDDDDDD&8??: NM:i:2 IH:i:4 HI:i:4
SRR959590.491 147 chr10 36684912 61 48S50M = 36684807 -155 GGCTCCTTTATGGTAGATGGATAAAATAAAAAAAAAAGAAATAAATAAAATAGCATATAAATAAGATAAAGATAAGGCAGGAACGAAATGAAATAAGA DDA?5(CCCC@:5(@ADDCCDCCAEC?CFJIJJIHCIEJIIEIHGBBHIGCH??IIIGIIGIJJIJIJJJJGJJJJJJIIGGHJIHHHHHFFFFFCCC NM:i:1 IH:i:4 HI:i:1
SRR959590.491 401 chr11 34180774 61 48S50M = 34180669 -155 GGCTCCTTTATGGTAGATGGATAAAATAAAAAAAAAAGAAATAAATAAAATAGCATATAAATAAGATAAAGATAAGGCAGGAACGAAATGAAATAAGA DDA?5(CCCC@:5(@ADDCCDCCAEC?CFJIJJIHCIEJIIEIHGBBHIGCH??IIIGIIGIJJIJIJJJJGJJJJJJIIGGHJIHHHHHFFFFFCCC NM:i:1 IH:i:4 HI:i:2
SRR959590.491 401 chr8 46854688 61 48S50M = 46854583 -155 GGCTCCTTTATGGTAGATGGATAAAATAAAAAAAAAAGAAATAAATAAAATAGCATATAAATAAGATAAAGATAAGGCAGGAACGAAATGAAATAAGA DDA?5(CCCC@:5(@ADDCCDCCAEC?CFJIJJIHCIEJIIEIHGBBHIGCH??IIIGIIGIJJIJIJJJJGJJJJJJIIGGHJIHHHHHFFFFFCCC NM:i:1 IH:i:4 HI:i:3
SRR959590.491 401 chr8 85697683 61 48S50M = 85697578 -155 GGCTCCTTTATGGTAGATGGATAAAATAAAAAAAAAAGAAATAAATAAAATAGCATATAAATAAGATAAAGA
can anyone kindly help me how to sort out this problem?????
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blooming.daisy333 •
Where your sequencing is unstranded, mappers can still add a strand to a read if it crosses a splice site, and that splice site has a canonical splice site sequence - it can use this sequence to work out which way the RNA is going.
Mappers such as TopHat (which cufflinks was originally designed to work with) only accepted splice sites with one of the canonical splice sites (i'm guessing), thus all spliced reads would contain the XS tag. This is definately true for HiSat when you run it in cufflinks compatible mode.
My guess is that MapSplice is allowing reads without the canonical splice site, and thus there are some reads that it can't orientate, so it isn't adding a tag.
As for how to solve it.... you can either find a way to tell MapSplice not to map non-canonical splice sites, use another mapper or filter out reads with non-canonical splice sites. They are probably not real anyway. There is probably some samtools foo for this, but I would do it in python with pysam:
from pysam import AlignmentFile
inBam = AlignmentFile("mybam.bam")
outBam = AlignmentFile("mybam.filtered.bam", "wb", template=inbam)
for read in inbam:
# output all unspliced reads
if 'N' not in read.cigarstring:
outBam.write(read)
# read is spliced check for XS tag
if read.has_tag('XS'):
outBam.write(read)
outBam.close()
thank you so much for the kind help. i wonder when the filter for non canonical sites was off in MapSplice then why it has added those reads ??? I would try your script and will discus the output.
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...and remember that a 'blooming daisy' will eventually produce beautiful flowers and will stand on its own 2 feet.
ooooo thanks for your encouragement. always stay blessed..
Thank you so much for the guidance. I ll keep these points in mind.
Thank you so much for the guidance. I ll keep these points in mind.