Question: Paired-End Reads and Variant Calling
0
gravatar for ocortes
6 weeks ago by
ocortes0
ocortes0 wrote:

Dear colleagues,

I have the fastq files of paired end sequences of 42 animals but when I run bwa mem to generate the .sam files i did not get the correct output, it is empty.

bwa mem -M CHR6.fa file_1.fastq file_2.fastq > out_2R.sam.

Can anyone help me?

Thanks in advance

bwa sam fastq • 206 views
ADD COMMENTlink modified 6 weeks ago by h.mon31k • written 6 weeks ago by ocortes0

what are the error/messages displayed ?

ADD REPLYlink written 6 weeks ago by Pierre Lindenbaum130k

Why don't you also put in the command you used to index CHR6.fa, just to cover all the bases.

ADD REPLYlink written 6 weeks ago by swbarnes28.6k

the fastq files has only sequences of chromosome 6

bwa index -a bwtsw CHR6.fa  
java -jar picard.jar CreateSequenceDictionary R= CHR6.fa O= CHR6.dict  
samtools faidx CHR6.fa

thanksĀ”

ADD REPLYlink modified 6 weeks ago by h.mon31k • written 6 weeks ago by ocortes0
1

By the way, it's always a bad idea to align the read on only one chromosome rather than on the whole reference.

ADD REPLYlink written 6 weeks ago by Pierre Lindenbaum130k

The commands run, this is the message that I can see in the screen, but the output file only has three lines

screen output

ADD REPLYlink written 6 weeks ago by ocortes0

The image doesn't work. Can't you just cut and paste the output?

ADD REPLYlink written 6 weeks ago by swbarnes28.6k

sorry, the link to the image:

bwa output

ADD REPLYlink modified 6 weeks ago by h.mon31k • written 6 weeks ago by ocortes0

That's an error. Did these fastq come from the sequencer like that or did you do so something to it?

ADD REPLYlink written 6 weeks ago by WouterDeCoster44k

This is a problem with the way your reads are ordered in the two fastq. Both files should have the read same names and in the very same order.

ADD REPLYlink written 6 weeks ago by Pierre Lindenbaum130k
3
gravatar for h.mon
6 weeks ago by
h.mon31k
Brazil
h.mon31k wrote:

Both your previous thread (BBmerge output question) and this one suffer from the same problem: the question leaves out important information. In both questions, the error probably arose from a previous step in your workflow (processing of the raw fastq files), but as you didn't provide details, the errors were not easily found - and still haven't been. You will have to describe in detail - providing all commands - how the fastq files have been processed so far. Also, stating your final goals may help - for example, in the other thread, it was pointed out merging reads is not necessary for calling SNPs.

You said you are a beginner, so, for the time being, the best course of action would be to follow one tutorial, from start to end. Try to find a tutorial which clearly states the versions of the programs being used, then you can use the same versions. Of course, it would be better if the tutorial is recent. You can certainly read several tutorials, but don't mix commands from them at first. If you do so, there will be plenty of additional hurdles along the way, such as different tutorials using different versions of the same programs, with incompatible command-line formats between these versions.

ADD COMMENTlink written 6 weeks ago by h.mon31k
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