Bowtie + samtools problem

[scami]

Hi guys

I generated an alignment with bowtie asking for a sam format. Everything went nice and smooth and got the file paired_mapped.sam. After this I wanted to use the samtools package and therefore, after installing, the first thing I did was to use the samtools import command. After elaborating for a couple of hours I received the following error message:

<code>
sam_header_line_parse] expected '@XY', got [@ILLUMINA-C3C24B_0047:1:1:1052:1111#0 77 * 0 0 * *0 0 TTTATCTAATAAATGCATCCNTTCCAGAAGTCGGGGTTTGTTGCACGTATTAGCTCTAGAATTACTACGGTTANC bbbbbbbbbbbcbbb`````Ebbbb`````bbbbbbabbbbbbbbbbbbbbbbbbba`bbcbbb`bb`b`BBBBB XM:i:0]
Hint: The header tags must be tab-separated.
[sam_header_read2] 33 sequences loaded.
<\code>



the first lines of my sam files are

[code]
@HD VN:1.0 SO:unsorted
@SQ SN:chr1 LN:23037639
@SQ SN:chr1_random LN:568933
@SQ SN:chr2 LN:18779844
@SQ SN:chr3 LN:19341862
@SQ SN:chr3_random LN:1220746
@SQ SN:chr4 LN:23867706
@SQ SN:chr4_random LN:76237
@SQ SN:chr5 LN:25021643
@SQ SN:chr5_random LN:421237
@SQ SN:chr6 LN:21508407
@SQ SN:chr7 LN:21026613
@SQ SN:chr7_random LN:1447032
@SQ SN:chr8 LN:22385789
@SQ SN:chr9 LN:23006712
@SQ SN:chr9_random LN:487831
@SQ SN:chr10 LN:18140952
@SQ SN:chr10_random LN:789605
@SQ SN:chr11 LN:19818926
@SQ SN:chr11_random LN:282498
@SQ SN:chr12 LN:22702307
@SQ SN:chr12_random LN:1566225
@SQ SN:chr13 LN:24396255
@SQ SN:chr13_random LN:3268264
@SQ SN:chr14 LN:30274277
@SQ SN:chr15 LN:20304914
@SQ SN:chr16 LN:22053297
@SQ SN:chr16_random LN:740079
@SQ SN:chr17 LN:17126926
@SQ SN:chr17_random LN:829735
@SQ SN:chr18 LN:29360087
@SQ SN:chr18_random LN:5170003
@SQ SN:chr19 LN:24021853
@SQ SN:chrUn LN:43154196
@PG ID:Bowtie VN:0.12.5 CL:"dir"
@ILLUMINA-C3C24B_0047:1:1:1052:1111#0 77 * 0 0 * * 0 0TTTATCTAATAAATGCATCCNTTCCAGAAGTCGGGGTTTGTTGCACGTATTAGCTCTAGAATTACTACGGTTANC bbbbbbbbbbbcbbb`````Ebbbb`````bbbbbbabbbbbbbbbbbbbbbbbbba`bbcbbb`bb`b`BBBBB XM:i:0
[\code]



I do not understand what is going wrong, the field are separated by tabs.
Any idea please?

thanks a lot for your help

S

[maubp]

I'd guess there is a missing new line in this bit:

@ILLUMINA-C3C24B_0047:1:1:1052:1111#0

Either that or your readnames start with @ which is not allowed in SAM format?

[scami]

Hi there

thanks for your reply. I gave a better look at the sam format and the number of "new line" should be correct. You are right when you say that the @ symbol can not be used in the reads lines. At the moment I am removing them, but I don't understand how come bowtie does not do this by its own when output a Sam format. I mean it gets in input Fastaq files which of course have the @symbol, and then when it outputs the sam file does not remove such symbol.... sound a bit wierd.....

[maubp]

What version of bowtie are you using? It sounds like a bug if all the read names in the SAM output have a @ at the start.

[scami]

I am using version 0.12.5. May be I did something wrong with the command (I am just beginning to play around with these software..... and with this topic actually.... ) The command I used is the following:
./bowtie-0.12.5/bowtie -v 2 -k 5 --best --fr -p 8 -I 100 -S --solexa1.3-quals ./indexes/riferimento_pinot --12 exp_47_s_1.fastq_bowtie_pe,exp_47_s_2.fastq_bowtie_pe,exp_47_s_3.fastq_bowtie_pe paired_end.map

is there anything wrong?

thanks a lot for your help

[maubp]

Well bowtie 0.12.5 is several months out of date, currently at 0.12.7, but the release notes don't mention any SAM output bug fixes:

http://bowtie-bio.sourceforge.net/index.shtml

Could you post the first few reads of your FASTQ files?

P.S. Use the [ code ] and [ /code ] tags to display it nicely on the forum (but without the spaces I have put round the square brackets). If you use the advanced editor then this can be accessed via the # toolbar button. Your original post tried <code> ... <\code> and [ code ] ... [ \code ] which are both wrong - use the other slash.

[scami]

Hi,

I used a script to covert my fastaq files in order to be used with bowtie. I had paired mates in one file and I used the 12 flag in bowtie. In according with the manual the input file should have been in a TAB separated text format:

<r>   Comma-separated list of files containing a mix of unpaired and paired-end reads in Tab-delimited format. Tab-delimited format is a 1-read-per-line format where unpaired reads consist of a read name, sequence and quality string each separated by tabs. A paired-end read consists of a read name, sequnce of the #1 mate, quality values of the #1 mate, sequence of the #2 mate, and quality values of the #2 mate separated by tabs. Quality values can be expressed using any of the scales supported in FASTQ files. Reads may be a mix of different lengths and paired-end and unpaired reads may be intermingled in the same file. If - is specified, bowtie will read the Tab-delimited reads from the "standard in" filehandle.

Therefore I used a script to convert the fastaq file which generated the following output:

@ILLUMINA-C3C24B_0047:1:1:1052:12086#0/1	TTCCGCGTCCTGACCTCCCCNGTTCAAGTAAGGCAACAACTACATATCCATCCTCTGCGTTAATCCATGTtaant	bbbbbbbbbbbbbbbbbbbaDaaaa```a`bb`bbbbbbbbbbbbbbbbb`bbbbbb_bbbb]bb_cbaaBBBBB	aaatttnggggtnagcaagtaacatacctaaagttgaaacataggnnancnancgagccacannnnannngnnnn	_____]E]]]NNEOO[[ZYV_____________\_________BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB

.... all fields are TAB separated. Considering your observations I think I should modify the script to avoid to output the '@' symbol in the bowtie input file. What do you think?

thanks a lot

[maubp]

Therefore I used a script to convert the fastaq file which generated the following output:

...

.... all fields are TAB separated. Considering your observations I think I should modify the script to avoid to output the '@' symbol in the bowtie input file. What do you think

Yes, remove the @ from your tabular output - you are telling Bowtie the read names all start with an @ character, so it is putting this in the SAM output (which is invalid).

Ideally I think you should also report this as a bug in Bowtie - arguably it should check the readnames don't start with @ when writing SAM format.

In FASTQ files, the @ is just a record marker - not part of the read name.