why Samtools Flagstat Results showing very less reads mapped

Question

[inayat45shaikh]

Hello group,

I am trying to get the percentage of reads mapped to reference using samtools. I used the command samtools flagstat sequence.bam, but the result I am getting is a bit worse than I was expecting, as it shows only 34% of reads mapped.

438974667 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 duplicates
151842378 + 0 mapped (34.59%:-nan%)
0 + 0 paired in sequencing
0 + 0 read1
0 + 0 read2
0 + 0 properly paired (-nan%:-nan%)
0 + 0 with itself and mate mapped
0 + 0 singletons (-nan%:-nan%)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

I wanted to know why I am getting this result, and where have I done mistake and how can fix this problem.

Thanks in advance

[Philipp Bayer]

I need to know more to be able to answer that -

What program did you use for alignments? Which settings, what insert size did you use? How complete is your reference? Is it the same species, or are you using a different reference genome? Is it mate-paired data? Did you have a look at the quality of your reads using FastQC?

[inayat45shaikh]

Hi Philipp,

i have a single-end data and i want to map this to its assembled contigs to assess assembly quality and calling variants. i used BWA for alignment.

[Devon Ryan]

How well reads will map to assembled contigs will depend entirely on the quality of the assembly. My guess is that this assembly is a bit lacking.

Answer 1

[Devon Ryan]

The results that it shows are simply the metrics that exist. If you want a higher alignment rate, then perhaps tweaking the aligner settings or changing how reads are pre-processed would be useful. Samtools just reports what's in the BAM file, nothing more.