running DEXSeq with hisat2 created bam files

[frymor]

Hi,

I am trying to run DEXSeq with bame files made by the hisat2 mapper. I have created the gff file according to the manual. I have seen that the chromosome names are different from the bam files, so I have awk-modified them. now they both look similar.

I have attached here the first 1000 rows of the gff file as well as 400 of the bam file from hisat2.
here are the first few rows of both files:

[B]gff[/B]:
1       dexseq_prepare_annotation.py    aggregate_gene  11869   14409   .       +       .       gene_id "ENSG00000223972.5"
1       dexseq_prepare_annotation.py    exonic_part     11869   12009   .       +       .       transcripts "ENST00000456328.2"; exonic_part_number "001"; gene_id "ENSG00000223972.5"
1       dexseq_prepare_annotation.py    exonic_part     12010   12057   .       +       .       transcripts "ENST00000450305.2+ENST00000456328.2"; exonic_part_number "002"; gene_id "ENSG00000223972.5"
1       dexseq_prepare_annotation.py    exonic_part     12058   12178   .       +       .       transcripts "ENST00000456328.2"; exonic_part_number "003"; gene_id "ENSG00000223972.5"
1       dexseq_prepare_annotation.py    exonic_part     12179   12227   .       +       .       transcripts "ENST00000450305.2+ENST00000456328.2"; exonic_part_number "004"; gene_id "ENSG00000223972.5"
1       dexseq_prepare_annotation.py    exonic_part     12613   12697   .       +       .       transcripts "ENST00000450305.2+ENST00000456328.2"; exonic_part_number "005"; gene_id "ENSG00000223972.5"

[B]bam[/B]:
SOLEXA3_1:8:54:9864:6930/2      433     1       10536   1       50M     16      90128665        0       TACCCCCGAAATCTGTGCAGAGGAGAACGCAGCTCCGCCCTCGCGGTGCT      ##################################################      AS:i:-4 ZS:i:-4 XN:i:0  XM:i:2  XO:i:0  XG:i:0NM:i:2   MD:Z:4A19C25    YT:Z:UP NH:i:8
SOLEXA3_1:8:96:12216:10104/2    401     1       10536   1       50M     =       532371  0       TACCACCGAAATCTGTGCAGAGGAGAACGCAGCTCCGCCCTCGCGGTGCT      BBCB<>=@:=)BCCCBCCDC@@@@:C>C@CCCCCC@CCCBACCBCCCCCC      AS:i:-4 ZS:i:-4 XN:i:0  XM:i:1  XO:i:0  XG:i:0  NM:i:1MD:Z:24C25       YT:Z:UP NH:i:6
SOLEXA3_1:8:38:7825:12181/2     177     1       10537   1       50M     2       61785065        0       ACCACCGAAATCTGTGCAGAGGAGAACGCAGCTCCGCCCTCGCGGTGCTC      #########B-A6=0;6<:6:66:*;34:.ABBA?7>AA=ABC?C@DBCC      AS:i:-4 ZS:i:-4 XN:i:0  XM:i:1  XO:i:0  XG:i:0NM:i:1   MD:Z:23C26      YT:Z:UP NH:i:6
SOLEXA3_1:8:102:12459:18067/1   337     1       10540   1       50M     19      238009  0       ACCGAAATCTGTGCAGAGGAGAACGCAGCTCCGCCCTCGCGGTGCTCTCC      BCCCCCC@CCCCCCCCCCCCCCCCCCCBCCCCCCCCCCCCCCCCCCCCCC      AS:i:-5 ZS:i:-5 XN:i:0  XM:i:1  XO:i:0  XG:i:0  NM:i:1MD:Z:20C29       YT:Z:UP NH:i:4
SOLEXA3_1:8:63:4265:15899/2     153     1       10565   255     50M     =       10565   0       CAGCTCCGCCCTCGCGGTGCTCTCCGGGTCTGTGCTGAGGAGAACGCAAC      A@<BB=B@BB2/5..(*0*/>CB>@BBC@B??;?9?C=C=<<78:;<;;<      AS:i:0  ZS:i:-2 XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0MD:Z:50  YT:Z:UP NH:i:1

When running the command:

python ~/R/x86_64-pc-linux-gnu-library/3.2/DEXSeq/python_scripts/dexseq_count.py -p yes gencode.v23.annotation.mod.gff $file DEXSeq_completeSample/$NEW_FILE.txt

I keep getting the following error message:

Traceback (most recent call last):
  File "/home/yeroslaviz/R/x86_64-pc-linux-gnu-library/3.2/DEXSeq/python_scripts/dexseq_count.py", line 215, in <module>
    for af, ar in HTSeq.pair_SAM_alignments( reader( sam_file ) ):
  File "/usr/local/lib/python2.7/dist-packages/HTSeq-0.6.1p1-py2.7-linux-x86_64.egg/HTSeq/__init__.py", line 601, in pair_SAM_alignments
    for almnt in alignments:
  File "/usr/local/lib/python2.7/dist-packages/HTSeq-0.6.1p1-py2.7-linux-x86_64.egg/HTSeq/__init__.py", line 536, in __iter__
    algnt = SAM_Alignment.from_SAM_line( line )
  File "_HTSeq.pyx", line 1276, in HTSeq._HTSeq.SAM_Alignment.from_SAM_line (src/_HTSeq.c:24611)
ValueError: ('SAM line does not contain at least 11 tab-delimited fields.', 'line 1 of file hisat2.bamFiles/610W1AAXX_8.sorted.bam')

the first line of the bam file looks like that:

SOLEXA3_1:8:54:9864:6930/2      433     1       10536   1       50M     16      90128665        0       TACCCCCGAAATCTGTGCAGAGGAGAACGCAGCTCCGCCCTCGCGGTGCT      ##################################################      AS:i:-4 ZS:i:-4 XN:i:0  XM:i:2  XO:i:0  XG:i:0NM:i:2   MD:Z:4A19C25    YT:Z:UP NH:i:8

Has anyone ever tried to run DEXSeq with the hisat2 output?
Is there a difference to the "standard" sam/bam format?

I would appreciate any help solving this error message.

thanks
Assa