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All the alignments in your SAM file excerpt have an alignment quality (5th column) of zero but, by default, dexseq-count.py only counts read with an alignment quality of at least 10. Try to find out why they are all zero, and if this is just a bogus output of your aligner, use the option '-a 0' to set the minimum quality to 0. -
let me give an example.
A couple of lines from my gtf file shows coordinates of a gene and its first exon
If i want to view all reads mapping to the first exon, i can use samtoolsCode:chr1 dexseq_prepare_annotation.py aggregate_gene 300422454 300435350 . + . gene_id "GRMZM2G077596" chr1 dexseq_prepare_annotation.py exonic_part 300422454 300422756 . + . transcripts "GRMZM2G077596_T01"; exonic_part_number "001"; gene_id "GRMZM2G077596"
Any help will be greatly appreciatedCode:samtools view input.bam chr1:300422454-300422756
this is the output for the previous command
Code:GALZUI2_0001:2:103:1190:211#0/1 0 chr1 300422484 0 36M * 0 0 CTTCTTTTGTTCTTTAATTTGGTTCGTACGTACAAG HHHHFEHCCHCCHHHHHGHHG<BEGHCFHHHHHCHH MD:Z:36 RG:Z:s_2_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:2:6:220:1972#0/1 0 chr1 300422515 0 36M * 0 0 ACAAGACTTCTCGGATCACTCGTCTTCTTTGATTGC HHHHHHHHHFHHHHHHHHHHHHHHHHGHHHHHHHHG MD:Z:36 RG:Z:s_2_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:2:42:1567:1468#0/1 0 chr1 300422526 0 36M * 0 0 CGGATCACTCGTCTTCTTTGATTGCATCATCGAGAC HHHHHHHHHHIHHHHHHHHHHHHHHGHHHHHHHHEH MD:Z:36 RG:Z:s_2_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:2:120:1465:1034#0/1 0 chr1 300422541 0 36M * 0 0 CTTTGATTGCATCATCGAGACCTGCATTTTCCCTTC CDCCC;GDGG;GGGGGG7G<GGGGGGGGGGGGGGGG MD:Z:36 RG:Z:s_2_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:2:11:502:602#0/1 0 chr1 300422554 0 36M * 0 0 ATCGAGACCTGCATTTTCCCTTCCAAATTCGTCACT HHHFHHHHHFHHFHHHHHHHHHDHHHEEHHCEHHHH MD:Z:36 RG:Z:s_2_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:2:99:1503:859#0/1 16 chr1 300422569 0 36M * 0 0 TTCCCTTCCAAATTCGTCACTCACTCTGGTTGGCCG FHHGHCHDGHHHGHCHGHGHHHHHEHHHHHBGGGBG MD:Z:36 RG:Z:s_2_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:2:118:626:857#0/1 16 chr1 300422569 0 36M * 0 0 TTCCCTTCCAAATTCGTCACTCACTCTGGTTGGCCG H?DDHH<GHHGHHHHD;HEHHDHHHGHHHHGHHDHH MD:Z:36 RG:Z:s_2_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:2:59:1772:1903#0/1 0 chr1 300422580 0 36M * 0 0 ATTCGTCACTCACTCTGGTTGGCCGCCTTCTGTCTT FHHHHHHGHHHDHHHHHDHHHHHHEHHHHHHHGHHH MD:Z:36 RG:Z:s_2_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:2:75:95:1416#0/1 16 chr1 300422586 0 1S35M * 0 0 ACACTCACTCTGGTTGGCCGCCTTATGTCTTCTGAT ##########B?288<10*7=?..'7?C3CBD?DHF MD:Z:23X11 RG:Z:s_2_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:2:18:1001:1651#0/1 0 chr1 300422587 0 36M * 0 0 ACTCACTCTGGTTGGCCGCCTTCTGTCTTCTGATCC HHHHHHHGHHHHHGEHHFHHHHHHHHHHHHHHHHHH MD:Z:36 RG:Z:s_2_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:2:54:199:1717#0/1 16 chr1 300422600 0 36M * 0 0 GGCCGCCTTCTGTCTTCTGATCCAATCCGGTTGAAA DHHFHIHHHHHHEHHHGHHDHHHFHHHHHDHHHHHH MD:Z:36 RG:Z:s_2_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:2:27:889:358#0/1 16 chr1 300422647 0 1S35M * 0 0 TCTTCCAGCAAGATCTGGCACATAAGGAGAATCGGC GHHH=HHHHHHHHFH=HHFEHHHHHHHGHHHHHHHC MD:Z:35 RG:Z:s_2_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:2:37:242:1709#0/1 16 chr1 300422679 0 36M * 0 0 GGCAAGAACCATTCTGCAAATGAGGCCGGATACGCG HFHHHHHHHHHHHHGHHHHHHHHHHHHHHHHHHHHH MD:Z:36 RG:Z:s_2_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:2:78:1327:822#0/1 16 chr1 300422679 0 36M * 0 0 GGCAAGAACCATTCTGCAAATGAGGCCGGATACGCG HHHHHHHFHGHHHHFHHHGHHHHHHHHHHHHHHHHH MD:Z:36 RG:Z:s_2_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:2:106:745:1364#0/1 16 chr1 300422680 0 1S35M * 0 0 TGCAAGAACCATTCTGCAAATGAGGCCGGATACGCG HHHHHHHEHHHHH8HHHHHHHBHHHHHEGHIHIGHH MD:Z:35 RG:Z:s_2_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:2:112:510:330#0/1 16 chr1 300422712 0 36M * 0 0 GCGGCTTGAATCGGCGGTGTTCCAGCTCACCCCGAC HHHGHIGFHIHFHGHIHHHHGHEHHHGEHGHHHHHH MD:Z:36 RG:Z:s_2_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:2:76:91:1422#0/1 0 chr1 300422734 0 23M357N13M * 0 0 CAGCTCACCCCGACCCGCACCAGGTGTGATTTAGTT HHHHHHHGIHHGHGHGHHHHHBHH=HHFEHHHEHHH MD:Z:36 RG:Z:s_2_sequence.txt.gz IH:i:1 NH:i:1 YS:A:+ GALZUI2_0001:2:36:34:147#0/1 0 chr1 300422741 0 16M357N20M * 0 0 CCCCGACCCGCACCAGGTGTGATTTAGTTGTGGTGG HHHHHGHHHHHHHIHHHHHHHHIHHHHHHHHHHHHG MD:Z:36 RG:Z:s_2_sequence.txt.gz IH:i:1 NH:i:1 YS:A:+
But in the output from
Code:python dexseq_count.py zea_mays.AGPv2.62_mod.gff sorted_sam.sam counts_exons.txt
and then searching for the gene gives
Code:grep GRMZM2G077596 counts_exons.txt GRMZM2G077596:001 0 GRMZM2G077596:002 0 GRMZM2G077596:003 0 GRMZM2G077596:004 0 GRMZM2G077596:005 0 GRMZM2G077596:006 0 GRMZM2G077596:007 0 GRMZM2G077596:008 0 GRMZM2G077596:009 0 GRMZM2G077596:010 0 GRMZM2G077596:011 0 GRMZM2G077596:012 0 GRMZM2G077596:013 0
Last edited by alpesh; 06-18-2013, 10:28 PM.Leave a comment:
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hi dpryan, i have to use AGPv2.62 annotation,,,the my gtf file has chr1,chr2 etc., when you say none of the reads are paired end, it is because of the column 2 flags being 16 (reverse strand), 0(forward strand) and 4(unmapped) ?Leave a comment:
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Well, I don't know about the AGPv2.62 version, but at least in the AGPv3.18 available from ensembl there are no chromosomes/contigs with names like chr1, chr2, etc. That could certainly result in 0 counts for everything (though probably some warnings too). Perhaps try opening the coordinate-sorted bam file in IGV or something similar and see if there's any obvious visual reason to get 0 counts.Leave a comment:
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Here is the command I used, all the exons (for 30k genes) come up with a 0 number in the output if nothing is specified about paired-eCode:python dexseq_count.py zea_mays.AGPv2.62.gff sorted_samfile.sam exon_counts.txt
top of output file
Code:AC147602.5_FG004:001 0 AC147602.5_FG004:002 0 AC147602.5_FG004:003 0 AC147602.5_FG004:004 0 AC147602.5_FG004:005 0 AC148152.3_FG001:001 0 AC148152.3_FG001:002 0 AC148152.3_FG001:003 0 AC148152.3_FG001:004 0 AC148152.3_FG002:001 0
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None of your reads are mapped as pair-end (at least of the reads you used in your example). Why you're still not getting any counts when not specifying that you have paired-end reads I don't know. Can you post the exact command that you're using?Leave a comment:
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getting dexseq_count.py to work
Hi,
Can you help me in this? I have paired end reads in sam format, and I have followed all steps to create the sorted sam file according to the passilla tutorial. I have successfully created the maize gtf file using the first python script. This is how my sam file looks like,
Code:GALZUI2_0001:8:100:0:142#0/1 4 * 0 0 * * 0 0 NCCTGGTGGAGACCGGAGGAGCCTCGGCAGAGATCG #0--011+++858::7=386>?;=:9?9==8??### RG:Z:s_8_sequence.txt.gz IH:i:0 NH:i:0 YU:Z:unmapped GALZUI2_0001:8:100:0:1654#0/1 16 chr1 128019411 0 35M1S * 0 0 TTTTCTCTGTTGATATTTCAATCTTCTTCCTCAGAN FFFFFFFFFFFFFF=FFFFFF??===44574,,00# MD:Z:35 RG:Z:s_8_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:8:100:0:1914#0/1 0 chr5 84824688 0 1S24M240N11M * 0 0 NTTTGCAGCTGATGCTGAGAGCAAGATTGTCCCTGC #################################### MD:Z:8X26 RG:Z:s_8_sequence.txt.gz IH:i:1 NH:i:1 YS:A:+ GALZUI2_0001:8:100:0:1953#0/1 4 * 0 0 * * 0 0 NCATAAACGATGCCGACCAGGGATCAGCGAGATCGG #################################### RG:Z:s_8_sequence.txt.gz IH:i:0 NH:i:0 YU:Z:unmapped GALZUI2_0001:8:100:0:1957#0/1 0 chr8 166503511 0 1S35M * 0 0 NACAAGGTAGGCCTCAGCCGCCTCCTGCAGCGCGGA #################################### MD:Z:35 RG:Z:s_8_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:8:100:0:2018#0/1 0 chr8 48995004 0 1S35M * 0 0 NAAGGGTATAACATCTCTGATGTTCTCCATTCCGGT #/-,+//.,,9:???==<<=FFF<<=:=?=881:8= MD:Z:35 RG:Z:s_8_sequence.txt.gz IH:i:2 NH:i:2 YR:i:1 GALZUI2_0001:8:100:0:2018#0/1 0 chr8 49024165 0 1S35M * 0 0 NAAGGGTATAACATCTCTGATGTTCTCCATTCCGGT #/-,+//.,,9:???==<<=FFF<<=:=?=881:8= MD:Z:35 RG:Z:s_8_sequence.txt.gz IH:i:2 NH:i:2 YR:i:1 GALZUI2_0001:8:100:0:287#0/1 4 * 0 0 * * 0 0 NCGGGGTTTCTTATGCGTGGATCCGGGAGATCGGAA #################################### RG:Z:s_8_sequence.txt.gz IH:i:0 NH:i:0 YU:Z:unmapped GALZUI2_0001:8:100:0:356#0/1 0 chr8 175216115 0 1S35M * 0 0 NTCGAATACATGTCCTCTCTTCTGGTTCAGAACACC #################################### MD:Z:35 RG:Z:s_8_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:8:100:0:362#0/1 4 * 0 0 * * 0 0 NAACAGCATGGATCCACCTTTTTCCCAACCTTTGAG #*+('(++)+8::::1:60:==:<;=====FFF;FF RG:Z:s_8_sequence.txt.gz IH:i:0 NH:i:0 YU:Z:unmapped GALZUI2_0001:8:100:0:434#0/1 16 chr1 300430123 0 35M1S * 0 0 ACCTTACTCTATGCAAGGCATGCCTTACTATCCTGN #################################### MD:Z:35 RG:Z:s_8_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:8:100:0:596#0/1 0 chr2 104752145 0 1S35M * 0 0 NGATGTGGTTGCGAAGAATGGCATGACGATGGTTGA #################################### MD:Z:35 RG:Z:s_8_sequence.txt.gz IH:i:4 NH:i:4 YR:i:1 GALZUI2_0001:8:100:0:596#0/1 0 chr4 68856217 0 1S35M * 0 0 NGATGTGGTTGCGAAGAATGGCATGACGATGGTTGA #################################### MD:Z:35 RG:Z:s_8_sequence.txt.gz IH:i:4 NH:i:4 YR:i:1 GALZUI2_0001:8:100:0:596#0/1 0 chr5 193863017 0 1S35M * 0 0 NGATGTGGTTGCGAAGAATGGCATGACGATGGTTGA #################################### MD:Z:35 RG:Z:s_8_sequence.txt.gz IH:i:4 NH:i:4 YR:i:1 GALZUI2_0001:8:100:0:596#0/1 0 chr9 90763379 0 1S35M * 0 0 NGATGTGGTTGCGAAGAATGGCATGACGATGGTTGA #################################### MD:Z:35 RG:Z:s_8_sequence.txt.gz IH:i:4 NH:i:4 YR:i:1 GALZUI2_0001:8:100:1000:1005#0/1 16 chr1 31201451 0 36M * 0 0 AAATATGGCACATATCAGGTGAACAGTGACCAAAAC =886A<A?8)CEB=.CFEF88>CEHHDHBHEEEGEG MD:Z:36 RG:Z:s_8_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:8:100:1000:1011#0/1 4 * 0 0 * * 0 0 GCTACATCGACCTTTCGAAGCGTCGCGAGATCGGAA AGEFBAEGGGECFHHHGCHDHHHFFHDHFHHH?HBC RG:Z:s_8_sequence.txt.gz IH:i:0 NH:i:0 YU:Z:unmapped GALZUI2_0001:8:100:1000:1039#0/1 4 * 0 0 * * 0 0 TCTCATGTGATGAGAAGTAGAACTAGTGGAGAGATC FFFFF:FFEF4EBFGFE>FFEFFFFFFFFFFFFFFF RG:Z:s_8_sequence.txt.gz IH:i:0 NH:i:0 YU:Z:unmapped GALZUI2_0001:8:100:1000:1073#0/1 4 * 0 0 * * 0 0 ACCAGAGCCTGTCCGTGGATGGGACCGGAGATCGGA HHEEHCHEHEGFHHEE=H/HEH9DHFEEF?=@GA## RG:Z:s_8_sequence.txt.gz IH:i:0 NH:i:0 YU:Z:unmapped GALZUI2_0001:8:100:1000:1076#0/1 4 * 0 0 * * 0 0 GACAAGTTGGCCCACCAGAATATGAGCCTACAGGAA GH1HHHEHHHHHHHFFFF6FFFFFFFF?FFF-FEF< RG:Z:s_8_sequence.txt.gz IH:i:0 NH:i:0 YU:Z:unmapped GALZUI2_0001:8:100:1000:1089#0/1 4 * 0 0 * * 0 0 GCTCGATGGCGGATGAAAATCAGGCAGATCGGAAGA HHCHHEHDHHF@EFF6=C;AE?BFFEE?EFBDA?FF RG:Z:s_8_sequence.txt.gz IH:i:0 NH:i:0 YU:Z:unmapped GALZUI2_0001:8:100:1000:1099#0/1 16 chr9 15614973 0 2S34M * 0 0 TCTCAACGTTTGAAGAAAAACCGTGAGATATACCGG 8HHHGHGE=BCHHHHFGDB6EHFGEHHBDHD?GFFG MD:Z:34 RG:Z:s_8_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:8:100:1000:11#0/1 4 * 0 0 * * 0 0 GTGGAGACGCAGGCGTGGAAGAGATCGGAAGAGCGG AAA@>6>@/<DG;C?GGGDGGCGGCEEGEGGGGAGG RG:Z:s_8_sequence.txt.gz IH:i:0 NH:i:0 YU:Z:unmapped GALZUI2_0001:8:100:1000:1145#0/1 0 chr6 147883848 0 32M4S * 0 0 ATTGTCGGCAACGGCGGGAAGCACCGCTGCCCCGCC FGFG>DDHDHGEFADA#################### MD:Z:32 RG:Z:s_8_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:8:100:1000:1173#0/1 16 chr1 269036723 0 36M * 0 0 TGTCAGGGACATGAAGGAGAAGCTCGCCTACATTGC FD?BDFCC?6<<CC@>C6:1AGGEAG???7@AAC@= MD:Z:36 RG:Z:s_8_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:8:100:1000:1195#0/1 16 chr3 135445725 0 36M * 0 0 ACGGCGCCTGCCGCAAAGATCATAGATACAGTTGGA ###@?=6.GGCCGGGFG6GDGGGGGCGGCGGGGGGG MD:Z:36 RG:Z:s_8_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:8:100:1000:121#0/1 0 chr3 3313715 0 34M2S * 0 0 GCCTCACTGAATATTCCAGCAGCTGTTGGCTGGGAG HHHHHHHHHHIHHHHHHHHHHHHHHHHHHHHHHFGH MD:Z:34 RG:Z:s_8_sequence.txt.gz IH:i:3 NH:i:3 YR:i:1 GALZUI2_0001:8:100:1000:121#0/1 0 chr8 25015676 0 34M2S * 0 0 GCCTCACTGAATATTCCAGCAGCTGTTGGCTGGGAG HHHHHHHHHHIHHHHHHHHHHHHHHHHHHHHHHFGH MD:Z:34 RG:Z:s_8_sequence.txt.gz IH:i:3 NH:i:3 YR:i:1 GALZUI2_0001:8:100:1000:121#0/1 16 chr4 63782093 0 2S34M * 0 0 CTCCCAGCCAACAGCTGCTGGAATATTCAGTGAGGC HGFHHHHHHHHHHHHHHHHHHHHHHIHHHHHHHHHH MD:Z:34 RG:Z:s_8_sequence.txt.gz IH:i:3 NH:i:3 YR:i:1 GALZUI2_0001:8:100:1000:1217#0/1 16 chr10 68420921 0 36M * 0 0 ACCTGAAGAGTGTTAGGGAATTGATCTACAAAAGAG 50?EEEGEGEGEEBGED??@;CDGGDC?CCBBD=DD MD:Z:36 RG:Z:s_8_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:8:100:1000:1249#0/1 0 chr1 222562953 0 36M * 0 0 AAAGGATGAGACCAGAGAATCATAGCAATCAGCTCA HHGCHH@HHGDHHEHAGE5GHHHHHHF7HHHHEHHE MD:Z:36 RG:Z:s_8_sequence.txt.gz IH:i:3 NH:i:3 YR:i:1 GALZUI2_0001:8:100:1000:1249#0/1 16 chr2 41634471 0 36M * 0 0 TGAGCTGATTGCTATGATTCTCTGGTCTCATCCTTT EHHEHHHH7FHHHHHHG5EGAHEHHDGHH@HHCGHH MD:Z:36 RG:Z:s_8_sequence.txt.gz IH:i:3 NH:i:3 YR:i:1 GALZUI2_0001:8:100:1000:1249#0/1 16 chr5 161906550 0 36M * 0 0 TGAGCTGATTGCTATGATTCTCTGGTCTCATCCTTT EHHEHHHH7FHHHHHHG5EGAHEHHDGHH@HHCGHH MD:Z:36 RG:Z:s_8_sequence.txt.gz IH:i:3 NH:i:3 YR:i:1 GALZUI2_0001:8:100:1000:1371#0/1 0 chr10 89544264 0 35M1S * 0 0 CTTATGCTTCACTTTTACTATAGGCTCAGAACTTTT GGGFAHHHHHHHHHHHHGGHHHHGHHHGHHHHHHHE MD:Z:35 RG:Z:s_8_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:8:100:1000:1465#0/1 4 * 0 0 * * 0 0 CGGTTCAGCAGGAATGCCGAGATCGGAAGAGCGGTT >BBEEBEEEF3FCC@FFFEFFFFFBFEFFF=FBFFE RG:Z:s_8_sequence.txt.gz IH:i:0 NH:i:0 YU:Z:unmapped GALZUI2_0001:8:100:1000:1489#0/1 16 chr2 176558351 0 36M * 0 0 CGTGACAAGTGCAGGAAACAAACCACTGAAAAGAAT @=DA@6F<<@>F?DDG>7C=?6==4;A@6ADEBEBE MD:Z:36 RG:Z:s_8_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:8:100:1000:1496#0/1 4 * 0 0 * * 0 0 CCAGATCAGCGTCGACTCATTTCGGGAGATCGGAAG GHEHHHHHFHGGGG??AG=GEGGGGGGGDGBGEGGE RG:Z:s_8_sequence.txt.gz IH:i:0 NH:i:0 YU:Z:unmapped GALZUI2_0001:8:100:1000:1536#0/1 16 chr9 94009423 0 36M * 0 0 AGTCAAATGTAACAACTGGTTTTAGCTTGATCTCTT FHHHHHHGHHHEHHHHFHHHHHHHGHHHHHHHHHHH MD:Z:36 RG:Z:s_8_sequence.txt.gz IH:i:1 NH:i:1 GALZUI2_0001:8:100:1000:1555#0/1 4 * 0 0 * * 0 0 CGTCGTTGGGGTAGTAGACGGCAGATCGGAAGAGCG FFBBFFHHHFHHHFEHHHHAHHHHHFDHHHFHHGHH RG:Z:s_8_sequence.txt.gz IH:i:0 NH:i:0 YU:Z:unmapped GALZUI2_0001:8:100:1000:1559#0/1 4 * 0 0 * * 0 0 GCAGATTTCACCAAGTGTTGGATTGTTCAGATCGAA HHDGHFHHHHHFH@B?HGHCHHHHE:HHFFHEHHFH RG:Z:s_8_sequence.txt.gz IH:i:0 NH:i:0 YU:Z:unmapped
I`m getting the following error. Though the reads are paired-end, the program does not recognize it as paired end. How do I get the program to run? If I dont specify any parameter (not use --pair=yes), it gives me an output with all 0 counts.
Code:File "dexseq_count.py", line 132, in <module> for af, ar in HTSeq.pair_SAM_alignments( HTSeq.SAM_Reader( sam_file ) ): File "/usr/lib64/python2.6/site-packages/HTSeq-0.5.4p3-py2.6-linux-x86_64.egg/HTSeq/__init__.py", line 612, in pair_SAM_alignments raise ValueError, "'pair_alignments' needs a sequence of paired-end alignments" ValueError: 'pair_alignments' needs a sequence of paired-end alignments
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