Question: problem with BWA alignment
0
gravatar for blur
23 months ago by
blur100
European Union
blur100 wrote:

Hi,

I am trying to align PE reads using bwa and something isn't working... I tried bwa mem ref.fa read1.fq read2.fq > file.sam but I get an error

[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] skip orientation FR as there are not enough pairs
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs

I also tried using SAMPE: i.e. I created two seperate sai files and ran

bwa sampe ref.fa read1.sai read2.sai read1.fq read2.fq > file.sam

and the file I end up with has like two reads in it...

can someone point out what I am doing wrong? Thanks!

bwa rna-seq • 1.2k views
ADD COMMENTlink modified 23 months ago by shwethacm200 • written 23 months ago by blur100
2

This is not an error, it's an information. Convert created SAM file to BAM file, sort it with samtools and check alignments statistics by samtools flagstat. You will see then how many reads were aligned etc. Best, Agata

ADD REPLYlink written 23 months ago by agata88770
1

No worries.

check this out: Error while running BWA mem

ADD REPLYlink written 23 months ago by 2nelly150
1

It's possible that your read pairing is broken, or that you're using the wrong reference so nothing is aligning, or the quality is too low for anything to align. Lots of possibilities - I suggest you start by stating what organism you are working with and BLASTing or Sketching the reads to see what they are, and running FastQC and posting the results. Also, the first 10 read headers from each file would be helpful, as well as any information you have about what platform they came from and how they have been preprocessed.

ADD REPLYlink modified 23 months ago • written 23 months ago by Brian Bushnell16k

I added markup to your post for increased readability. You can do this by selecting the text and clicking the 101010 button. When you compose or edit a post that button is in your toolbar, see image below:

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ADD REPLYlink written 23 months ago by WouterDeCoster39k
0
gravatar for shwethacm
23 months ago by
shwethacm200
Seattle, WA
shwethacm200 wrote:

What you are seeing is not really an error. For some reason, most of your reads are aligning very poorly to your reference. Are you aligning to the wrong reference? This could result in the kind of results you are seeing. Did you preprocess your fastq files (trim adapters, remove linkers?). This could sometimes mess with alignments.

ADD COMMENTlink modified 23 months ago • written 23 months ago by shwethacm200

Thanks for all the help - I think the clean-up process I am using is what's ruining my pairs... It a specific protocol that has a collapse for identical reads, and I think this is how pairs get lost (i.e. if I have 10 reads identical in each fastq file, the ones which survive are not the original pairs) If I were to align each read separately and than put them into a single file, will I be loosing too much info? each read is 100bp. (btw the reference genome is correct (I double checked).)

Thanks!

ADD REPLYlink written 23 months ago by blur100
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