PacBio raw .bam file

[anotherSAM]

I have just received data from my first PacBio sequencing operation and was unaware that the new output format was in .bam file for raw reads, as they are calling the 'better fastq'.
I am using a pipeline, beginning with canu, which requires a fastq file however when using both samtools and bamtools to generate a fastq file from the bam file, the quality row just contains exclamation marks

samtools bam2fq data.bam > data.fastq
bamtools convert -format fastq -in data.bam -out data.fastq
e.g.
@read1
ATGCATGCAGCTGATGCTAGCATGCTACTAGTCGATCGTAGCTAGTCGATCGATGCTAGCATCGATGCTAGCTAGTCGATGCTAGCTGCGTAGCTGATGATGCTAGTCGACTGATACGAT
+
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!


Additional output files were a bam.pbi, an xml and a fasta file

Does anyone know how to handle the raw read bam files in order to generate fastq files with the appropriate quality score?

[Brian Bushnell]

It's possible that the bam file does not contain quality scores. Try using samtools view to convert the bam to sam, and post the first couple reads from it...

[GenoMax]

Have you tried bam2fastx from PacBio?

[sklages]

What platform? At least for Sequel data the subread-bam/fastq files do not have quality values associated to the bases. So there is a dummy value for each base, zero, "!".

[anotherSAM]

head -n1 test.sam 
m54072_170901_055052/5112430/0_2238	4	*	0	255	*	*	0	0	TTCCGGGGATGGGGGGTCTTGGTATTGGACATCTATATGGTTCCTTTCCACTAAACTTGAGGCATCAGGCCTGTTTGGACCGGAGTACGTAAATTTCGTTTTCGTTATTTTCGATCCATGGCTCATTCTTCGTTGGCGCTTTTCTATCAAGAGATGAGGAACCAGCTCTACTCTATGTT	!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!	bc:B:S,1,1	bq:i:85	cx:i:10	ip:B:C,1,61,22,90,8,8,72,3,7,40,9,68,4,15,8,12,72,34,156,61,10,3,50,9,70,74,16,17,2,28,6,38,60,13,11,13,122,14,42,70,7,12,14,32,25,21,38,14,7,8,34,2,1,7,5,12,60,61,6,5,8,9,52,3,16,182,7,86,44,15,56	np:i:1	pw:B:C,7,5,13,13,2,11,2,5,6,10,3,6,47,13,24,41,7,6,7,6,39,34,12,44,5,11,18,21,8,15,14,7,7,2,6,5,8,30,28,3,4,2,22,15,12,13,4,15,2,12,10,7,8,17,5,9,2,22,26,24,7,13,11,6,9,6,6,10,3,11,3,3,14,6,4,17,10     qe:i:2238	qs:i:0	rq:f:0.8	sn:B:f,7.81349,14.5671,6.42793,10.4147	zm:i:5112430RG:Z:25f8c430

Brian, This is what the respective sam file looks like (i removed the majority of the nucleotides, numeric values and exclamation points for better clarity).
Sklages, This is with sequel data, however if it is true that the bam files do not have a quality score, where is it? From what I had read, the new bam output was meant to replace fastq.
Genomax, I followed the link for installing the bam2fastx library tool, which considering no error messages occurred I thought had succeeded, but when I went to use the bam2fastq command, it was unavailable.

Thank you all for your replies

[GenoMax]

Genomax, I followed the link for installing the bam2fastx library tool, which considering no error messages occurred I thought had succeeded, but when I went to use the bam2fastq command, it was unavailable.

Did you check the local directory where you did the install for one called bam2fastx or something similar. The executables should be inside that directory, if the build was successful.

[sklages]

Sklages, This is with sequel data, however if it is true that the bam files do not have a quality score, where is it? From what I had read, the new bam output was meant to replace fastq.

Which BAM are you reading? "subreads"? PacBio stated back in february:

"The subread-bam/fastq files do not have quality values associated to the bases. In fact none of our SMRT Analysis tools use or need them (e.g. the much improved CCS2 algorithm doesn't need the qualities anymore), and because there is no "placeholder" quality value as far as I know (like N for bases), the qualities in the BAM files are set to the lowest value "!"."


And as our current BAM files also have "!" values, I assume that this has not changed (yet).

[GenoMax]

Interesting that PacBio chose to set the quality values to lowest setting rather than highest (or somewhere north of Q30).

[sklages]

That what my first thought too ... it is not a very smart idea choosing basically a "junk value" :-)

[anotherSAM]

Genomax, I managed to get the bam2fastx tools working but they provided the same information as samtools and bamtools.
But as we have come to understand this is of no fault of the tools and is actually due to the output.
Sklages, So in this way the new bam files are only able to be analysed using SMRT software?

[sklages]

It's probably a good idea to adjust the quality dummy values to something "more or less good", e.g. 30 or more before using third-party tools ..

[GenoMax]

You could use reformat.sh from BBMap suite to set a fake Q score of 30 for each base like this.

reformat.sh in=pbio_input.bam out=stdout.fa | reformat.sh in=stdin.fa out=new.fq.gz qfake=30

[anotherSAM]

I will most likely substitute the quality scores, but how are they written in PacBio data.
But it is coded scores in fastq so 30 corresponds to something like >
This should work. It will mean canu is unable to trim reads and increase the real quality but I will also run it against illumina data to correct errors afterwards.

Thank you all

UPDATE: With substitution of a qscore of 30 for each base canu assembly has issues and produces a large number of contigs ~130. Possibly has issues in ranking overlap probabilities.

[Lsterck]

Hi,

interesting approach, I might give that a try as well.

I was just wondering why canu would have more trouble with all qscores put to 30 than when all were still 0 ?
Also, canu accepts both fasta as fastq as input (or even a mixture) might it then not simply ignore any quality score present?

[gringer]

As fas as I know, canu also doesn't use quality scores for assembly. By default it does its own error correction based on an all-vs-all read mapping, and produces corrected FASTA files (i.e. without quality scores) as intermediate data files prior to assembly.