bam to fastq conversion, number of reads do not match

Question

[Anushka]

Hello, I am trying to convert some publically available .bam files to the fastq format, using picard tool function SamToFastq as following:

 $ java -Xmx4g  -jar picard.jar SamToFastq NON_PF=true INPUT=input.bam F=input_1.fastq.gz F2=input_2.fastq.gz FU=unpaired_input.fastq.gz

The resulting fastq files have lesser number of reads than original bam file. I am checking like this:

Using samtools view input.bam | wc -l resulted into 62193989

While,zcat input_1.fastq.gz | wc -l is reporting 103572960 (51786480)

Why is there are less number of reads in the fastq file? I have tried to with UNPAIRED_FASTQ=File option in picard tools, which is reporting zero reads.

I would appreciated if someone could explain why this is happening? Either I am trying with wrong approach of making a correspondence between above numbers or is there something going wrong during conversion. Which is the best way to check whether the .bam to .fastq conversion went well?

[ATpoint]

Do not use wc -l with samtools. Use samtools view -c in.bam, which is much faster. Be also aware that a fastq contains 4 rows per read. Specifically to your problem, be sure to use INCLUDE_NON_PRIMARY_ALIGNMENTS=true as Pierre suggested to include non-primary alignments. After doing that, count again.

Answer 1

[Pierre Lindenbaum]

Why is there are less number of reads in the fastq file?

because the the bam might contains some secondary alignments and supplementary alignments.

[Anushka]

Thank you so much Pierre. When I tried with samtools view -F 256 input.bam | wc -l then it returned me the exact value which matches the fastq.