Samtools Flagstat Comments

Question

[santos48]

Hi, I am trying to solve total alignments :0 results of featureCounts this problem. Now I checked my bam files with that command samtools flagstat .bam. What do you think about these results? Is this possible?

11214947 + 0 in total (QC-passed reads + QC-failed reads)
5640993 + 0 secondary
108597 + 0 supplementary
0 + 0 duplicates
11214947 + 0 mapped (100.00% : N/A)
0 + 0 paired in sequencing
0 + 0 read1
0 + 0 read2
0 + 0 properly paired (N/A : N/A)
0 + 0 with itself and mate mapped
0 + 0 singletons (N/A : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

[GenoMax]

You did not respond to my comment here: Salmon Quant /SubRead featureCounts Results Total Aligments:0

You likely have reads that are overlapping multiple features and thus are likely not being counted. You can turn on -M multi-mapping switch for featureCounts to test the other possibility that they are mapping to more than one location (multi-mappers) and are thus not being counted. You can see that in the secondary/supplementary alignment numbers. Over 50% of your reads are multi-mapping.

Since we don't have access to your data this is about the best we can do to assist.

[santos48]

Hey, thank you for the answer. I tried different functions such as --largestOverlap, -M, -O but they increased only other bam files it still total alignment:0. I tried extracting the region chrX(example) from the first bam file in the results. featureCounts can not count that bam file when I dont extract

     Total alignments : 239085                                               ||
||    Successfully assigned alignments : 72251 (30.2%)

[santos48]

I guess the problems coming from the total sequencing, featureCount does not apply if sequence over some number.

[GenoMax]

I don't think that is the case. featureCounts has been used with millions of reads. You only have 240K, granted they are long reads. Then again featureCounts was not designed for long read alignments. Your data must have something in it that is causing this.

[santos48]

Do you have an idea for start to check?