Question: Questions about bam to fastq
0
gravatar for oghzzang
2.4 years ago by
oghzzang40
oghzzang40 wrote:

Dear Biostar users.

I wanna convert bam to fastq.

So I used "samtools bam2fq".

This is my script.

**fastq1=`echo $(basename ${input_file%%.gz.*})`**

**fastq2=`echo ${fastq1/R1/R2}`**

**samtools bam2fq $input/$input_file > $output_dir/${input_file}_fastq**

**cat $output_dir/${input_file}_fastq | grep '^@.*/1$' -A 3 > $fastq1**

**cat $output_dir/${input_file}_fastq | grep '^@.*/2$' -A 3 > $fastq2**

This is right?


And I wanna identify whether my bam file includes unmapped reads. so I run samtools flagstat.

This is the result. In this result, can i think that my bam file includes unmapped reads?

40304229 + 0 in total (QC-passed reads + QC-failed reads)

0 + 0 secondary

0 + 0 supplementary

0 + 0 duplicates

40304225 + 0 mapped (100.00% : N/A)

39496046 + 0 paired in sequencing

19799602 + 0 read1

19696444 + 0 read2

39219752 + 0 properly paired (99.30% : N/A)

39486133 + 0 with itself and mate mapped

9909 + 0 singletons (0.03% : N/A)

219437 + 0 with mate mapped to a different chr

219437 + 0 with mate mapped to a different chr (mapQ>=5)

Thank you so much.

question bam fastq samtools • 1.6k views
ADD COMMENTlink modified 2.4 years ago by 5heikki9.0k • written 2.4 years ago by oghzzang40

I don't completely understand your script since it appears to be circular. I am going to assume that you are feeling a BAM file to samtools bam2fq.

Considering that the total number of reads is almost identical to mapped (only 4 less) it looks like your BAM must have only mapped reads (or your dataset was almost entirely mapped, rare but possible).

ADD REPLYlink written 2.4 years ago by genomax91k

Dear genomax. Thanks for your reply.

My script is ..

samtools bam2fq [in.bam] > [out.fastq]

cat [out.fastq] | grep '^@.*/1$' -A 3 > [fastq.1]

cat [out.fastq]| grep '^@.*/2$' -A 3 > [fastq.2]

And as you said, my flagstat result looks like all reads are mapped.

You've been great help to me. Thank you.

ADD REPLYlink modified 2.4 years ago • written 2.4 years ago by oghzzang40
0
gravatar for 5heikki
2.4 years ago by
5heikki9.0k
Finland
5heikki9.0k wrote:

Using the program properly would save a lot of time..

Usage: samtools bam2fq [options...] <in.bam>
Options:
  -0 FILE   write paired reads flagged both or neither READ1 and READ2 to FILE
  -1 FILE   write paired reads flagged READ1 to FILE
  -2 FILE   write paired reads flagged READ2 to FILE
  -f INT    only include reads with all bits set in INT set in FLAG [0]
  -F INT    only include reads with none of the bits set in INT set in FLAG [0]
  -n        don't append /1 and /2 to the read name
  -O        output quality in the OQ tag if present
  -s FILE   write singleton reads to FILE [assume single-end]
  -t        copy RG, BC and QT tags to the FASTQ header line
  -v INT    default quality score if not given in file [1]
      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
      --reference FILE
               Reference sequence FASTA FILE [null]
ADD COMMENTlink modified 2.4 years ago • written 2.4 years ago by 5heikki9.0k

Dear 5heikki. Thank you so much.

But I can't find how to convert bam to fastq.1 and fastq.2 at a time using bam2fq. In other tutorials using bam2fq, bam is converted to fastq and separated to fastq1, fastq2. In fact, among samtools bam2fq options, i don't know whether "-1" option mean fastq1 file.

Do you mean "samtools bam2fq -1 [fastq1] -2 [fastq2] [in.bam] ?

I'm very much obliged to your comment.

ADD REPLYlink written 2.4 years ago by oghzzang40

Absolutely, although the options are listed, they are not really usable. samtools bam2fq -1 [fastq1] -2 [fastq2] [in.bam] this also did not work.

ADD REPLYlink written 18 days ago by hiraksarkar.cs0
0
gravatar for genomax
18 days ago by
genomax91k
United States
genomax91k wrote:

Now you can do samtools fastq -1 R1.fastq.gz -2 R2.fastq.gz in.bam with latest samtools (v.1.11). Be sure to name sort (samtools sort -n) your BAM file before you do the conversion. See samtools manual page for option information.

ADD COMMENTlink modified 18 days ago • written 18 days ago by genomax91k
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