Re: [Samtools-help] samtools - unaligned reads

[Shawn Y. <yos...@gm...>]

Hi Neel,
  To extract all unmapped reads from a sam file type 'samtools view -Shf 4
IN.sam > unaligned.sam'  To get all aligned reads type 'samtools view -ShF 4
IN.sam > Aligned.sam'.   Note in awk you can do "awk '$2==4' IN.sam >
unAligned.sam"  To get aligned reads do "awk '$2!=4' IN.sam > Aligned.sam".
NOTE!  the awk command only works for a FRAGMENT read data set NOT for
paired-end.  Use the samtools view command for paired-end (or mate-paired)
data.

  Shawn

P.S.  to save space you may want to work in a stream and store things in
.bam format.  For example you can do the same above by typing "samtools view
-hbf 4 IN.bam > unAligned.bam"  or the awk command "samtools view -h IN.bam
| awk '$1~"@" || $2==4' | samtools view -Shb - > unAligned.bam"   Note the
$1~"@" is there to make sure you keep the header of the sam file.

On Mon, Mar 14, 2011 at 5:47 PM, Neel Aluru <na...@wh...> wrote:

> Hello,
>
> This is my first post and I am a beginner to samtools. So, please bear with
> me.
>
> 1. I am seeing a discrepancy in the number of unaligned reads I am getting
> with two different methods (samtools flagstat and filtering with awk).
> According to flagstat function, I have a total of 8187768 reads and 4553610
> are mapped. I presume the remaining 3634158 reads are unmapped.
>
> $ samtools flagstat MH_0001aligned_sorted.bam
> 8187768 in total
> 0 QC failure
> 0 duplicates
> 4553610 mapped (55.61%)
> 0 paired in sequencing
> 0 read1
> 0 read2
> 0 properly paired (nan%)
> 0 with itself and mate mapped
> 0 singletons (nan%)
> 0 with mate mapped to a different chr
> 0 with mate mapped to a different chr (mapQ>=5)
>
> 2. I want to write the unmapped reads to a new file, so I am using awk to
> search and write them to a separate file ( I know you can do this with
> samtools view function, but I am unable to do it, see below). That gives me
> a total of 3633980 reads as unaligned. Somewhere along, I am loosing 178
> reads. I am not concerned about them but just for peace of mind, I want to
> know where they have gone.
>
> $awk '($6 == "*")' MH_0001aligned.sam | wc -l
>  3633980
>
> With flagstat function, I used indexed and sorted bam file, whereas with
> awk, I used aligned sam file. Could this explain the differences?
>
> 3. Also, I am having a hard time using options with samtools view for
> filtering the reads. How can I filter the reads using -F option if I want to
> filter unmapped reads?
> I tried $ samtools view - F U MH_0001aligned_sorted.bam | wc -l and didn't
> get any result. How do I specify it so that I can filter correctly.
>
> Any comments or suggestions to explain this will be highly appreciated.
>
> Thank you,
> Neel
>
> Neel Aluru
> Postdoctoral Scholar
> Biology Department
> Woods Hole Oceanographic Institution
> Woods Hole, MA 02543
> USA
> 508-289-3607
>
>
>
>
>
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