How to use Bowtie2 for mapping pair end reads and in what format reference genome should be used?

Question

[priya.saxena]

Hello Everyone

I am trying to map the pair end reads using Bowtie2, I have formatted the custom reference genome using the format given in link: https://galaxyproject.org/learn/custom-genomes/ (used normalized custom reference file).

There is no error while running the Bowtie2 tool but I am getting the mapping stats like:

Warning: skipping mate #2 of read 'A00572:261:HHN3FDSXY:1:1101:27380:18912 2:N:0:GTTCTTCT+AATACCTT' because length (1) <= # seed mismatches (0)
Warning: skipping mate #2 of read 'A00572:261:HHN3FDSXY:1:1101:27380:18912 2:N:0:GTTCTTCT+AATACCTT' because it was < 2 characters long
Warning: skipping mate #2 of read 'A00572:261:HHN3FDSXY:1:1101:19271:25723 2:N:0:GTTCTTCT+AATACCTT' because length (1) <= # seed mismatches (0)
Warning: skipping mate #2 of read 'A00572:261:HHN3FDSXY:1:1101:19271:25723 2:N:0:GTTCTTCT+AATACCTT' because it was < 2 characters long
Warning: skipping mate #2 of read 'A00572:261:HHN3FDSXY:1:1101:25617:29199 2:N:0:GTTCTTCT+AATACCTT' because length (1) <= # seed mismatches (0)

I am really confused what does it mean and where I am getting wrong. Can anyone please help me out in solving this issue?

Thank you

[GenoMax]

Looks like you have reads that have become really short after trimming. Such reads and their mates should be removed from the input files as they are likely too short to be useful.

That said if you are doing this in galaxy then I am not sure what option you would use. It will depend on the tool you used for the initial trimming.

[Ram]

As a general rule, warnings can be ignored. It definitely helps to understand where they're coming from though.

[priya.saxena]

Thank you so much

But i am getting this kind of warning in whole file.