Samtools flagstat number of reads do not match actual total number of reads.

Question

[lakhujanivijay]

samtools flagstat results:

some number + 0 in total (QC-passed reads + QC-failed reads)

my this "some number" does not match actual number of reads in my paired fastq file. Is this expected?

[lakhujanivijay]

Coming back to my post after quite sometime

This command should give the exact number of reads in your sample:

expr $(samtools view -f 4 your.bam -c) + $(samtools view -F 2308 your.bam -c)

Explanation

Count the number of unmapped reads

samtools view -f 4 your.bam -c ( -f : accept the reads with the flag 4 , this flag is for unmapped reads)

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Count of the number of reads which do not have flag 2308( -F : reject the reads with the flag 2308 , flag explanation in below image)

samtools view -F 2308 your.bam -c

IMAGE COURTESY : Explain SAM FLAGS

Answer 1

[Devon Ryan]

The number of reads reported is the number actually in the file. If your aligner produced secondary alignments then this will often be higher than the original number in the fastq files.

[igor]

Also, the aligner may throw out unaligned reads.

Answer 2

[dariober]

My 2p: It's good to keep in mind that SAM/BAM stores alignments not reads (in fact, sam stands for sequence alignment/map) even if unmapped reads can be present.

For this reason the simple question "how many reads have been aligned?" can be tricky to answer. A simple strategy is to count the reads that have not been aligned and get the difference with the raw read count from fastq. But if you want to know how many reads have been aligned with certain criteria (e.g. mapq > x, alignment score > y, properly paired etc) than you should consider also split reads.

I suspect the use of the word "read" in samtools flagstat causes a lot of misunderstandings in this respect.