sam2bam problem

[m_elena_bioinfo]

Dear users,
I have run bwa on microRNA reads from ABI solid and I generated a file aln.sam.
But when I try to import this file in samtools, the program returns me different errors.
I can't convert SAM to BAM.
I'm using samtools-0.1.7

if I run:
>samtools import reference.fasta.fai aln.sam prova.bam
the error is
[sam_header_read2] 735 sequences loaded.
Parse error at line 1: invalid CIGAR character
Aborted

If I run:
>samtools import reference.fasta aln.sam prova.bam
the error
[sam_header_read2] 1102 sequences loaded.
Segmentation fault

With:
>samtools view aln.sam prova.bam
[bam_header_read] EOF marker is absent.
[main_samview] fail to read the header.

>samtools view -S aln.sam -o prova.bam
[samopen] no @SQ lines in the header.
[sam_read1] missing header? Abort!

>samtools view -S aln.sam prova.bam
[samopen] no @SQ lines in the header.
[main_samview] random alignment retrieval only works for indexed BAM files.

I can only think that the problem is the header of sam file.
But what can I do?

Thanx!

[dawe]

I usually convert sam files like this:

samtools view -bt reference.fa filein.sam | samtools sort - fileout

of course the sort process is optional. Note that fileout has no extension as it is automagically appended by samtools. reference.fa has a separate reference.fa.fai file.

d

[m_elena_bioinfo]

Hi dawe, thank you very much.
But the program doesn't run and the error this time is:

[sam_header_read2] 735 sequences loaded.
[sam_read1] reference 'hsa-mir-182' is recognized as '*'.
Parse error at line 1: invalid CIGAR character
Segmentation fault

[dawe]

I had similar issues when the sam file was not properly generated and the name of the matched chromosome was not in the reference file... which bwa and samtools version are you using?

[m_elena_bioinfo]

I'm usign bwa-0.5.4 and samtools-0.1.7!

[ramouz87]

sam2bam issue with maq

Hi,
I'm also having a problem converting sam2bam from a maq map file
seems that maq2sam-long did not add @ headers so sam file are generated :
HWI-EA332_8_80_1554_696#CAGTNN 89 GL000207.1 136 13 75M * 0 0 AGAGTAGTCAATAGGATAGATGAACGTACGTTGTATAAAAATTGTATTATCTAAAACTTCACAGAGAAAATAGGA %%%%%%%%%%%%%%%%%%%%%%%%%%%%88*=AA;>A12(>*>CCA+ACA?A?
BCAAABCCCCCCCCCCB>BCA4 MF:i:64 AM:i:0 SM:i:13 NM:i:15 UQ:i:87 H0:i:2 H1:i:0
HWI-EA332_8_80_1554_696#CAGTNN 149 GL000207.1 161 0 * * 0 0 GCGCTGCAGAAGTGGACGGAATCCAGATAAGTATTCTGCTAGGACAGGGG %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% MF:i:192
HWI-EA332_8_26_749_1572#CCCCNN 81 GL000207.1 795 75 75M * 0 3125 GGCATGGTTGCTGTGGCTATGAAGGGACTCATTTGAAGGGGCCATGGAAGAGCTGGCAGTATTATGATAGCAGAT ;156<3<7)/(9A@@32A7;@::A@67?><@?9@B??BBB>::<1@BA@BAB;
6@B=@BBBBB@9A@BBCABCBB MF:i:4 AM:i:69 SM:i:75 NM:i:0 UQ:i:0 H0:i:1 H1:i:0
HWI-EA332_8_73_1194_791#CCCCNN 81 GL000207.1 795 78 75M * 0 3125 GGCATGGTTGCTGTGGCTATGAAGGGACTCATTTGAAGGGGCCATGGAAGAGCTGGCAGTATTATGATAGCAGAT 7=665<;@A@=AB>@A?B@BA=8B@B6@B:8B@AB;?BBBA87.4@B2<C=B9
ABB==B>@>6=BB=>>A>=B=B MF:i:4 AM:i:63 SM:i:78 NM:i:0 UQ:i:0 H0:i:1 H1:i:0
HWI-EA332_8_96_43_1791#CCCCNN 81 GL000207.1 795 66 75M * 0 3125 GGCATGGTTGCTGTGGCTATGAAGGGACTCATTTGAAGGGGCCATGGAAGAGCTGGCAGTATTATGATAGCAGAT %%%%%%%=76.<=97@614;<=?8<?85958960<6;@@@@836?A<<=A=B=
@BA==B>A9@@:@@A?B@@BBA MF:i:4 AM:i:47 SM:i:66 NM:i:0 UQ:i:0 H0:i:1 H1:i:0

but when using:
samtools view -Sxhut ~/genomes/Homo_sapiens.GRCh37.56.dna.toplevel.fa -o maq.bam maq.sam
get a segmentation fault :
[sam_header_read2] 23923109 sequences loaded.
Segmentation fault

I'm using samtools 1.7 and maq 0.7.1
Anyone faced such case ?


Thanks
Ramzi

[m_elena_bioinfo]

BWA parameter for microRNA

Hi NGS users,
could anyone suggest me the best parameters for BWA for alignign reads (35bp SOLiD single-read) for microRNA samples?
I want to align the reads vs the human genome but i have some problem.

For the ALN step, which parameters I have to use?
Thanx a lot,
have a good weekend,
ME