[meher]

Just post the SAM file, so we can see what's wrong.

Here are the first few lines of sam file:

@HD VN:1.0 SO:coordinate
@SQ SN:gi|17737322|ref|chrMT| LN:16727
@SQ SN:gi|357579592|ref|chrX| LN:123869142
@SQ SN:gi|357579593|ref|chr38| LN:23914537
@SQ SN:gi|357579594|ref|chr37| LN:30902991
@SQ SN:gi|357579595|ref|chr36| LN:30810995
@SQ SN:gi|357579596|ref|chr35| LN:26524999
@SQ SN:gi|357579597|ref|chr34| LN:42124431
@SQ SN:gi|357579598|ref|chr33| LN:31377067
@SQ SN:gi|357579599|ref|chr32| LN:38810281
@SQ SN:gi|357579600|ref|chr31| LN:39895921
@SQ SN:gi|357579601|ref|chr30| LN:40214260
@SQ SN:gi|357579602|ref|chr29| LN:41845238
@SQ SN:gi|357579603|ref|chr28| LN:41182112
@SQ SN:gi|357579604|ref|chr27| LN:45876710
@SQ SN:gi|357579605|ref|chr26| LN:38964690
@SQ SN:gi|357579606|ref|chr25| LN:51628933
@SQ SN:gi|357579607|ref|chr24| LN:47698779
@SQ SN:gi|357579608|ref|chr23| LN:52294480
@SQ SN:gi|357579609|ref|chr22| LN:61439934
@SQ SN:gi|357579610|ref|chr21| LN:50858623
@SQ SN:gi|357579611|ref|chr20| LN:58134056
@SQ SN:gi|357579612|ref|chr19| LN:53741614
@SQ SN:gi|357579613|ref|chr18| LN:55844845
@SQ SN:gi|357579614|ref|chr17| LN:64289059
@SQ SN:gi|357579615|ref|chr16| LN:59632846
@SQ SN:gi|357579616|ref|chr15| LN:64190966
@SQ SN:gi|357579617|ref|chr14| LN:60966679
@SQ SN:gi|357579618|ref|chr13| LN:63241923
@SQ SN:gi|357579619|ref|chr12| LN:72498081
@SQ SN:gi|357579620|ref|chr11| LN:74389097
@SQ SN:gi|357579621|ref|chr10| LN:69331447
@SQ SN:gi|357579622|ref|chr9| LN:61074082
@SQ SN:gi|357579623|ref|chr8| LN:74330416
@SQ SN:gi|357579624|ref|chr7| LN:80974532
@SQ SN:gi|357579625|ref|chr6| LN:77573801
@SQ SN:gi|357579626|ref|chr5| LN:88915250
@SQ SN:gi|357579627|ref|chr4| LN:88276631
@SQ SN:gi|357579628|ref|chr3| LN:91889043
@SQ SN:gi|357579629|ref|chr2| LN:85426708
@SQ SN:gi|357579630|ref|chr1| LN:122678785
@PG ID:TopHat VN:2.0.9 CL:/csc/lohi/dog_tools/tophat-2.0.9.Linux_x86_64/tophat -p 3 -o ./ /csc/lohi/canFam3_ref_dogData/ncbi_fasta_canFam3/canFam3 B00EJHW.t30l32.pair1.fastq.gz B00EJHW.t30l32.pair2.fastq.gz
HISEQ4_0112:3:1101:1044:57612#GCCAAT 89 gi|357579618|ref|chr13| 621671950 101M * 0 0 TGCACTTGTTGAACTGCTGAAACACAAGCCCAAGGCAACAGATGAACAACTGAAAACTGTTATGGGAGATTTTGGAGCCTTTGTAGAGAAGTGCTGCGCNG @>3CDDC@3@DBC>6;6CC@EEBA?EEGBIGHCGIGFF@BC:JJJIIHCGD4BDGIFIGIGHEDGCJJIJJJIIHJJJJJIJJJJJHGHHHFFDDA4#C AS:i:-1 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:99A1 YT:Z:UUNH:i:1
HISEQ4_0112:3:1101:1044:61660#GCCAAT 99 gi|357579610|ref|chr21| 328438950 6M1452N95M = 32845462 1652 CNCAAGAGTTTACTCTCCAAAGCTCGAGGAATCGATTCCAGCTCTGTTAAACTCCGAGGTGGTTCTTTATTCATGGATACAGAAAAATCAGGAAAAAGGGA C#1ADFFFHHHHHJJJJJJJJJJJJJJJJJIJJJJJJJJIIJJJJJJJJJJJJJJJHIJEHHHHHFFFFFFFFEEEEEEDDDDDDDDDDDDDDDDDDDDDD AS:i:-1 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:1C99 YT:Z:UU XS:A:+ NH:i:1


Should you require the whole sam file to be attached?

[Simon Anders]

Your chomosome names don't match. HTSeq-count cannot guess that "gi|357579630|ref|chr1|" in the SAM file is the same as "chr1" in the GFF file.

[meher]

Your chomosome names don't match. HTSeq-count cannot guess that "gi|357579630|ref|chr1|" in the SAM file is the same as "chr1" in the GFF file.

Thank you. As i thought both have the same notation i.e. chr1,chr2.. they are in sync with each other but that seems to be costly.