bbwrap.sh, is output OK?

[floem7]

Hi all,

I have a library sequenced two times in paierd-end mode.

I have used bbwrap.sh

~/bin/bbmap/bbwrap.sh -in1=hds1r1_09clean.fastq.gz,hds1r1_26clean.fastq.gz -in2=hds1r2_09clean.fastq.gz,hds1r2_26clean.fastq.gz path=/media/mj/c8e2ccd2-6313-4092-be34-46144891720f/agp_v5 unpigz=t pigz=t threads=24 -Xmx100g outm=s68_to_b73v5.bam showprogress=250000 statsfile=stats_s68_to_b73v5 covstats=covstats_s68_to_b73v5

If I understand correctly it is possible to provide paired files in -in and -in2

The procedure proceeded ok, like bbmap.sh, after processing the first pair it printed info, which I expected:

------------------ Results ------------------

Genome: 1
Key Length: 13
Max Indel: 16000
Minimum Score Ratio: 0.56
Mapping Mode: normal

Reads: 461959640
Mapped reads: 455488907
Mapped bases: 44824369462
Ref scaffolds: 685
Ref bases: 2182075994

Percent mapped: 98.599
Percent proper pairs: 80.240
Average coverage: 20.542
Average coverage with deletions: 27.430
Standard deviation: 111.219
Percent scaffolds with any coverage: 100.00
Percent of reference bases covered: 98.53

Total time: 393719.639 seconds.

And later:

Executing align2.BBMap [build=1, overwrite=true, fastareadlen=500, unpigz=t, pigz=t, threads=24, showprogress=250000, statsfile=stats_s68_to_b73v5, covstats=covstats_s68_to_b73v5, indexloaded=t, in=hds1r1_26clean.fastq.gz, in2=hds1r2_26clean.fastq.gz]
Version 38.90

Set threads to 24
Retaining first best site only for ambiguous mappings.
No output file.
Cleared Memory: 0.308 seconds.
Processing reads in paired-ended mode.
Started read stream.
Started 24 mapping threads.
....................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................Detecting finished threads: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23

------------------ Results ------------------

Genome: 1
Key Length: 13
Max Indel: 16000
Minimum Score Ratio: 0.56
Mapping Mode: normal

Total time: 381683.967 seconds.


I'm surprised to see "No output file." in the messages above. Also the entire output ended with "total time" and no statistics as for the first read-pair.

So in the end I wonder if my resulting file is ok (I used outm to get only mapped reads). I.e. does it contain mapped reads from all four files?

I don't want to test if, for example merged file from separate runs of bbmap for both pairs would give the same number of mapped reads. It would take too much time.
bbwrap seems ok, but from my point of view it is not enough documented.

[GenoMax]

You can't use `-in1=` those options are simply `in1=` and `in2=` etc. You should also use `out=` not `outm=` if you are writing an alignment file.

bbwrap.sh in1=read1.fq,singleton.fq in2=read2.fq,null out=mapped.sam append

[floem7]

Thanks,

so can I write:

bbwrap.sh in1=hds1r1_09clean.fastq.gz,hds1r1_26clean.fastq.gz in2=hds1r2_09clean.fastq.gz,hds1r2_26clean.fastq.gz ... out=s68_to_b73v5.bam

?

Why I have to use "out" but not "outm"? I want to retain only mapped reads, to keep my file as small as possible.

It is also interesting, why bbwrap haven't alert that options such as "-in1" are invalid. Some output file has been written but I don't know if it is valid. At least bamqc accepted that file. Nevertheless I'd rather want properly generated file, even if it means that I have to wait for it ten days.

[GenoMax]

That would be fine. Looks like you can use `outm=` if you need just mapped reads. I have normally used `out=` with `mappedonly=t` but those two look to be equivalent.

It should not take 10 days to complete an analysis unless you have a huge dataset.

[floem7]

Ok, thanks. I have huge dataset so I will have to wait for results.

[floem7]

So my mapping crashed. Here is my command:
~/bin/bbmap/bbwrap.sh in1=hds1r1_09clean.fastq.gz,hds1r1_26clean.fastq.gz in2=hds1r2_09clean.fastq.gz,hds1r2_26clean.fastq.gz path=/media/mj/c8e2ccd2-6313-4092-be34-46144891720f/agp_v5 unpigz=t pigz=t threads=24 -Xmx100g outm=s68_to_b73v5.bam append showprogress=250000 statsfile=stats_s68_to_b73v5 covstats=covstats_s68_to_b73v5 bs=bs.sh

Here is the error message

[E::sam_parse1] no SQ lines present in the header
samtools view: error reading file "-"
Exception in thread "Thread-46" java.lang.RuntimeException: java.io.IOException: Przerwany potok
at stream.ReadStreamByteWriter.run(ReadStreamByteWriter.java:32)
Caused by: java.io.IOException: Przerwany potok
at java.base/java.io.FileOutputStream.writeBytes(Native Method)
at java.base/java.io.FileOutputStream.write(FileOutputStream.java:354)
at java.base/java.io.BufferedOutputStream.write(BufferedOutputStream.java:123)
at stream.ReadStreamByteWriter.writeSam(ReadStreamByteWriter.java:659)
at stream.ReadStreamByteWriter.processJobs(ReadStreamByteWriter.java:93)
at stream.ReadStreamByteWriter.run2(ReadStreamByteWriter.java:42)
at stream.ReadStreamByteWriter.run(ReadStreamByteWriter.java:28)
Exception in thread "Thread-49" java.lang.RuntimeException: Writing to a terminated thread.
at stream.ConcurrentGenericReadOutputStream.write(ConcurrentGenericReadOutputStream.java:202)
at stream.ConcurrentGenericReadOutputStream.addDisordered(ConcurrentGenericReadOutputStream.java:197)
at stream.ConcurrentGenericReadOutputStream.add(ConcurrentGenericReadOutputStream.java:97)
at align2.AbstractMapThread.writeList(AbstractMapThread.java:625)
at align2.AbstractMapThread.run(AbstractMapThread.java:591)
MORE REPETITIONS OF SIMILAR BLOCKS

**************************************************************************
Warning! 24 mapping threads did not terminate normally.
Check the error log; the output may be corrupt or incomplete.
Please submit the full stderr output as a bug report, not just this message.
**************************************************************************

I don't have to necesarily use bbwrap, I just wanted to make mapping in elegant way.
So just to make sure, could I map first pair and second pair of read separately with bbmap and then merge the results (bam files)? I don't have time for more tries, I have been waiting five days for this error.

[floem7]

Ok, so I've found samtools merge.

[GenoMax]

You need to map paired end files together before merging the resulting alignment files using samtools merge.