[Useq-users] USeq issue with incorrect mate position information for paired RNASeq data

[David N. <Dav...@hc...>]

Hello Guorong,

Yes, looks like an issue.  Would you mind grepping your unprocessed alignment file for those read names so I can see what it started out as.

-cheers, D

P.S. We don't use HTSeq so it's not an issue.  The USeq apps hash on the read name after collecting intersecting reads for each exon so paired data is collapsed if present.  I wonder how HTSeq is doing it?  As a short term workaround, use the USeq DefinedRegionDifferentialSeq with the -t option and it will generate a table of hit counts for each gene and each sample. http://useq.sourceforge.net/cmdLnMenus.html#DefinedRegionDifferentialSeq



From: Guorong Xu <guo...@gm...<mailto:guo...@gm...>>
Date: Wed, 24 Oct 2012 13:18:51 -0500
To: David Nix <dav...@hc...<mailto:dav...@hc...>>
Subject: USeq issue

Hi David,

I found an issue from Useq SamTranscriptomParser for paired-end dataset. Please see the below two fragments (4 reads).

The mate positions of the first fragment are not correct, but the mate positions of the second fragment are correct.
When I looked into the original alignment file by novoalign, I found the first fragment was mapped on a junction region (junction library).
The mate position of these two reads are not changed when Useq converts the artificial chrom name to genomic chrom name.
That’s why the mate positions of the first fragment are very small (457 and 345).

Now, we cannot use HTSeq to calculate the counts information due to this incorrect format (mate position issue).
Do you have any suggestions on that?

Thanks,
Guorong

HWI-ST1189:59:C1305ACXX:5:1101:10001:63267    99    chr21    48064279    0    51M    =    457    0    GGGGTGAGCGTGCGGGCTGCTGTGGGTAC
ATTCCGGCAAACCATGTGGGGA    BCCFDFFFHHHHHJJJJJJJJJGIIJFHIJJJJJJJJJHHHFFFFFFEDDD    PG:Z:novoalignMPI    NH:i:12    HI:i:1    AM:i:70    NM:i:0    SM:i:70    GN:Z:
951    TN:Z:ENST00000440086    ZN:i:12    PQ:i:2    UQ:i:2    AS:i:2    ZS:Z:R
HWI-ST1189:59:C1305ACXX:5:1101:10001:63267    147    chr21    48064391    0    10M3969N41M    =    345    0    TGGAACTCTGAAACTCCACTT
GGAGATGTTGGCAGACCAGCCACGAACAAC    EHGHDJIJGGHH9GFHD:BIGIHGGGGFHGIHGEFBJJHHHHHFFFFFCCC    PG:Z:novoalignMPI    NH:i:12    HI:i:1    AM:i:70    NM:i:0    SM:i:
70    GN:Z:951    TN:Z:ENST00000440086    ZN:i:12    PQ:i:2    UQ:i:0    AS:i:0    XS:A:-    ZS:Z:R

HWI-ST1189:59:C1305ACXX:5:1101:10001:74618    99    chr3    185135345    0    51M    =    185135507    0    CTGGTTTCTTGGTAGCTGCTG
CCTTTTTTCCCACCAGAGGCTTCTTCTGCT    @@@FBDDEFFHHFHHIIJJIIIJIIJJIIGDHIJIGGGHIFJJGGIBG@EF    PG:Z:novoalignMPI    NH:i:6    HI:i:1    AM:i:70    NM:i:0    SM:i:
70    ZN:i:6    PQ:i:3    UQ:i:3    AS:i:3    ZS:Z:R
HWI-ST1189:59:C1305ACXX:5:1101:10001:74618    147    chr3    185135507    0    51M    =    185135345    0    CCTTATCCACCCGGAGCTTGT
GATTCCTGGCCTGGCGAAGAATGGTGTTCC    @EGGEGIGDIHAGJIDIIIGHABHIGGDDCGGEGGFHFFFHHHDDDFDC@@    PG:Z:novoalignMPI    NH:i:6    HI:i:1    AM:i:70    NM:i:0    SM:i:
70    ZN:i:6    PQ:i:3    UQ:i:0    AS:i:0    ZS:Z:R