WARNING: reads from the same pair were found not adjacent to each

Question

[Kurk]

WARNING: reads from the same pair were found not adjacent to each || || other in the input (due to read sorting by location or || || reporting of multi-mapping read pairs).

Good afternoon. Can I know the cause of this warning in my featurecounts? I used this command:

featureCounts -T 8 -t exon -g gene_id -a gtf -o outputfile.txt -s2 -p MaMock-A.bam MaMock-B.bam MaMock-C.bam Ma48hpi-A.bam Ma48hpi-C.bam Ma48hpi-D.bam

to run datasets. Thank you

Answer 1

[ATpoint]

featureCounts requires name-sorted BAM files for paired-end data. Yours is apparently not. No worries as the tool is smart and sorts it automatically so you don't have to do anything. That's why it's a warning rather than an error. Are assignment rates ok?

[Kurk]

Thank you for the reply. These are my final assignment rate. Is that okay?

BeMock-A.bam Total fragments : 30159776 Successfully assigned fragments : 21748618 (72.1%)

BeMock-B.bam Total fragments : 28230493 Successfully assigned fragments : 19530208 (69.2%)

BeMock-C.bam Total fragments : 25746301 Successfully assigned fragments : 17614143 (68.4%)

Be48hpi-B.bam Total fragments : 26418825 Successfully assigned fragments : 22121142 (83.7%)

Be48hpi-C.bam Total fragments : 23767292 Successfully assigned fragments : 18670971 (78.6%)

Be48hpi-E.bam Total fragments : 25047836 Successfully assigned fragments : 19753950 (78.9%)