[aforntacc]

ok i will have a look at the link and try the script.
yes i compiled bowtie on the VM but its 64bits
thanks a lot.

[jp.]

Hi GenoMax
May you please help me on Tophat2 insert size commands. I used defaults but 0TSS and others 0...
I posted here http://seqanswers.com/forums/showthr...098#post112098
Thank you

Looking at the time stamps in your log it appears that the error is thrown after ~18 h of run time. That is a pain ..

Can you try the script posted by Simon Andrews in post #8 to check for errors in your fastq files: http://seqanswers.com/forums/showthread.php?t=7784

Did you compile bowtie on this VM? Are you using 32-bit Ubuntu or 64-bit?

[aforntacc]

Hi all
please what insert size should i use for (-r / -- mate - inner - dist) am a bit confused
i ran bowtie2 on a subset of the reads to be able to have an idea of the insert size but i saw different valuse in the alignment (9th field of the sam output file) and now i dont know which to choose. here is the header. thanks in advance
HWI-ST365_0157:7:1101:1975:2074#GCGGTC 77 * 0 0 * * 0 0 CCCANCTTCACACTCAAAATTTGTTCTGTAGTGTTTGACCATACACAACTTTTGTTTCTTTTGTTACAAAAGTATTGTATAATTGGAACTAAACAAAGGC _bbeBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB YT:Z:UP

HWI-ST365_0157:7:1101:1975:2074#GCGGTC 141 * 0 0 * * 0 0 TCAAAACACATGCACCCACTAGCTTCCTTGGAAAAANA a__ecceeeggggfffdgggfgeghdegfghffebgBB YT:Z:UP

HWI-ST365_0157:7:1101:1991:2120#GCGGTC 83 gi|359484478|ref|XM_002281860.2| 952 42 100M = 888 -164 TGCTTCACGACTGAGTAGTGACGACAAACATGCACGTTGCAGAATGAAATGACTTAAATTACCCTTGTTTTTATATGATTTGGAGTTAATGTAATGGGTG cccabcaab`dbdccdeeeegeggdgfbhihiihhihhiihhhihiihiihihggfhhhdciiihiiiiiiiiiiiiiihiiiiiiigggggeeeeebbb AS:i:-6 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:73A26 YS:i:0 YT:Z:CP

HWI-ST365_0157:7:1101:1991:2120#GCGGTC 163 gi|359484478|ref|XM_002281860.2| 888 42 40M = 952 164 GTACTTCCTACCATATGCGATGGGCATTCTCGTCATTTTA aabeeeeegggggifhiiiiiihifgggfh[egfgfhiic AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:40YS:i:-6 YT:Z:CP

HWI-ST365_0157:7:1101:2000:2177#GCGGTC 77 * 0 0 * * 0 0 CGAGAATCGGCTGGGGGCTTGGGTGATGGTCACTTGTTTCCAGACGTCTTGCATTTTGTCTGCCATGGTTTTGGCAGGGATGGCTTGGAGACGCCGTTGG ^__cccc]ce[ceefhW^^bU_R^UHONIOIONNaSM\bS_ecHLZ_N\``dHVZ_Y^BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB YT:Z:UP

HWI-ST365_0157:7:1101:2000:2177#GCGGTC 141 * 0 0 * * 0 0 CGGATAAATCACATACAAAAGCAATGGCACAGCAATCAAGGNCNCTA a__ecccae]bg]c`dffha_dReed`R[bfH_aHPY^fgeBBBBBB YT:Z:UP

HWI-ST365_0157:7:1101:2242:2071#GCGGTC 83 gi|359490881|ref|XM_002277964.2| 1169 40 100M = 1075 -194 GCATCACAATCAAGCCCCAAACTGATCGATGGGTTTTCCCAGAAACCAACAGTGGCATCATTATTCTCGCTGAGGGACGACTGATGAACTTAGGANGTGC BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBc___ AS:i:-1 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:95T4 YS:i:-35 YT:Z:CP

HWI-ST365_0157:7:1101:2242:2071#GCGGTC 163 gi|359490881|ref|XM_002277964.2| 1075 40 100M = 1169 194 AAGCAGATGAAGAACAATGCAATTGTTTGTAACATTGGCCAGTTTGACAATGAGATTGGTATGGTTGGTTTTGAAACCTACCCTGGGGTTAAGGGCATCA a__`ccccagecgda`[`beccegdedfJ`dgf[d[I^BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB AS:i:-35 XN:i:0 XM:i:7 XO:i:0 XG:i:0 NM:i:7 MD:Z:41C16A4C5C1G14T6C6 YS:i:-1YT:Z:CP

HWI-ST365_0157:7:1101:2219:2076#GCGGGC 153 gi|225443436|ref|XM_002269813.1| 66 0 100M = 66 0 CGGGCCGGTCTGGACGTCCGCCCCCCCCCACGGCACCCAAAAGGAGCGCAACCAGGTCGATGAAGCTCGCCGGAGCTTCGCCGACCTCCGGTTCGAGAAG BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBa`[_WZRffaf`ac`dd^_ihhgggeaecaee_a_ AS:i:-45 XN:i:0 XM:i:9 XO:i:0 XG:i:0 NM:i:9 MD:Z:1C3A7G0A2T2A5A2T10G59 YT:Z:UP

HWI-ST365_0157:7:1101:2219:2076#GCGGGC 69 gi|225443436|ref|XM_002269813.1| 66 0 * = 66 0 CAGANAGACAGTTGAGAGTTGAAACTAAATTGTATAATGTGGAAGCTGAAGGTGGCCGAAGGGGACGATCCCTGGCTCAGGACGTTGAACGGCCACGGCG _bbeBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB YT:Z:UP
bilbo@ubuntu:/media/My Passport/Trimmomatic-0.30$

[mastal]

You need to ask the sequencing center that ran your samples what the average insert length of the libraries was.

[alyamahmoud]

I have the same error:
Error: Couldn't build bowtie index with err = 1

however, both fasta and gtf files identify chromosome as chr 1!!

head btaurus.fa
ENSBTAT00000006175 range=chr1:67082115-67116669 5'pad=0 3'pad=0 strand=- repeatMasking=none
TCTTGGCGAAGTGGTGCGTCGTGGACGGGTCGCTGATGAAGAAAGACACA
GTGTCCCCGCAGTCCCTGAGCTGAAAGGGGCCGGAGAGCGCCGGTATGGG
CTCCGAGCTGCCCGCACCTTGGCTGCTGCTCTTCACCTGGCTCCCAACAG
gtgagccatgggaaaatgggtcctagccaggcccaaggagagaggaagcc
attgttgcctggcctaacagagttggccgcaggcgggacagaggaggcct

head btaurus.gtf
chr1	bosTau6_ensGene	stop_codon	67082857	67082859	0.000000	-	.	gene_id "ENSBTAT00000006175"; transcript_id "ENSBTAT00000006175"; 
chr1	bosTau6_ensGene	CDS	67082860	67082898	0.000000	-	0	gene_id "ENSBTAT00000006175"; transcript_id "ENSBTAT00000006175"; 
chr1	bosTau6_ensGene	exon	67082115	67082898	0.000000	-	.	gene_id "ENSBTAT00000006175"; transcript_id "ENSBTAT00000006175"; 
chr1	bosTau6_ensGene	CDS	67087347	67088182	0.000000	-	2	gene_id "ENSBTAT00000006175"; transcript_id "ENSBTAT00000006175";

Any clue ?

thanks a lot

[GenoMax]

This is the reason I had suggested getting the pre-formatted genomes/indexes from iGenomes. The source fasta genome files do not have names (e.g. chr1) that match the annotation file. Your genome file is also not in the "FASTA format" (http://en.wikipedia.org/wiki/FASTA_format).

For example (minimal change to file header to FASTA format):

From

ENSBTAT00000006175 range=chr1:67082115-67116669 5'pad=0 3'pad=0 strand=- repeatMasking=none

To

>ENSBTAT00000006175 range=chr1:67082115-67116669 5'pad=0 3'pad=0 strand=- repeatMasking=none

Even with this change you will not be able to use the genome annotation file you have.

[alyamahmoud]

Hi GenoMax

Thanks a lot for your reply. you are true, I still have the same error and cannot generate bowtie indices so far. However, I cannot use the preformatted genomes/indices from iGenome package as bos taurus is not among them !! any other ideas ?

I tried gtf/seq formats from UCSC but wasn't successful either.

[GenoMax]

Bos taurus genome/indexes are available on this page: http://support.illumina.com/sequenci...e/igenome.ilmn See 4th row in the table.

Index files are not available standalone. They are part of a big download that contains genome sequence, indexes for bwa/bowtie and annotation. You will have to get the entire tarball ( *.gz file). All these files are synced (i.e. chromosome names will match across all files etc) to work together.

[alyamahmoud]

Thank you very much indeed, thanks a lot. I was only checking the iGenomes at the tophat/cufflinks web page. Thank you for the valuable info.

[justwalking]

hi, I hope this suggestion could be useful
gene ids in your btaurus.fa should be the same as "chr1" and so on

I have the same error:
Error: Couldn't build bowtie index with err = 1

however, both fasta and gtf files identify chromosome as chr 1!!

head btaurus.fa
ENSBTAT00000006175 range=chr1:67082115-67116669 5'pad=0 3'pad=0 strand=- repeatMasking=none
TCTTGGCGAAGTGGTGCGTCGTGGACGGGTCGCTGATGAAGAAAGACACA
GTGTCCCCGCAGTCCCTGAGCTGAAAGGGGCCGGAGAGCGCCGGTATGGG
CTCCGAGCTGCCCGCACCTTGGCTGCTGCTCTTCACCTGGCTCCCAACAG
gtgagccatgggaaaatgggtcctagccaggcccaaggagagaggaagcc
attgttgcctggcctaacagagttggccgcaggcgggacagaggaggcct

head btaurus.gtf
chr1	bosTau6_ensGene	stop_codon	67082857	67082859	0.000000	-	.	gene_id "ENSBTAT00000006175"; transcript_id "ENSBTAT00000006175"; 
chr1	bosTau6_ensGene	CDS	67082860	67082898	0.000000	-	0	gene_id "ENSBTAT00000006175"; transcript_id "ENSBTAT00000006175"; 
chr1	bosTau6_ensGene	exon	67082115	67082898	0.000000	-	.	gene_id "ENSBTAT00000006175"; transcript_id "ENSBTAT00000006175"; 
chr1	bosTau6_ensGene	CDS	67087347	67088182	0.000000	-	2	gene_id "ENSBTAT00000006175"; transcript_id "ENSBTAT00000006175";

Any clue ?

thanks a lot