Variable numbers of Reads. I'm lost



I have a question regarding the read count. After the sequencing, i found that i have 513k reads that were passed. But when i align (minimap2) and check how much reads i have in my bam file using Samtools, i found myself with 1.7M reads.
After that i was looking for the mapped reads only and i have 280k.
Than when i use featureCount to quantify and count my reads, it tells me that i have 1.7M reads, but assign only 268k reads.
Can you help me please ?

Another thing : I was also wondering how to quantify rRNAs in my sequencing using featureCount. I tried to retrieve all rRNAs feature in the count file generated by featureCount, but the number of reads aligning to the rRNAs is greater than the reads assigned by featureCount.

Thank you very much for your help!!







First, note that the number of reads is not the same concept as the number of alignments. One read may produce several alignments. The BAM file contains alignments not reads.

Run the:

samtools flagstat

command. It will describe your data in a report like:

31571 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
1571 + 0 supplementary
0 + 0 duplicates
31345 + 0 mapped (99.28% : N/A)
30000 + 0 paired in sequencing
15000 + 0 read1
15000 + 0 read2
29616 + 0 properly paired (98.72% : N/A)
29762 + 0 with itself and mate mapped
12 + 0 singletons (0.04% : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

it will show you how your alignments are distributed.

before adding your answer.

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