bwa-meth unrecognized reference name during sam_parse

Question

[bp22]

Dear all,

I have some Enzymatic methyl sequencing data that I am trying to align using bwa-meth (https://github.com/brentp/bwa-meth). However, I am encountering the following issue in parsing of the sam file that the aligner is generating when I try to convert the sam file to a bam file.

[W::sam_parse1] unrecognized reference name ""; treated as unmapped

This is the first time for me using bwa-meth tool and I am not able to solve this issue. I am running the following command as suggesting on the GitHub page mentioned above.

bwameth.py --reference ref_genome.fa Sample1_1.fastq.gz Sample1_2.fastq.gz -t 7 | samtools view -b - > sample1.bam

Any help is appreciated.

Thank you.

[GenoMax]

Do you have a blank fasta entry (without a name) in your reference?

[bp22]

Hi GenoMax

Many thanks for your response.

I am using the hg38 genome fasta file from GENCODE. Upon indexing the reference genome as suggested in bwa-meth:

bwameth.py index $REF

another file with $REF.bwameth.c2t is produced apart from the other indexing files. I do not file any blank fasta entry (without a name) in the original fasta file that I have downloaded from GENCODE. However, in the $REF.bwameth.c2t file the fasta entry has names changed from

>chr1 1

to

>rchr1 1

and this might be a reason for samtools not recognising the reference name as is mentioned in the message generated by the aligner. I am not yet sure but will get back once I have checked this after correcting the fasta entry name in the $REF.bwameth.c2t file.

Best, bp22

[GenoMax]

If the indexing process created the .c2t files then you should not edit/make any changes to to the files. The issue must lie some place else.

[TabeaK]

Have you been able to solve this? I am running exactly into the same issue using bwameth right now.

[bp22]

Hi,

I had the same issue while running bwameth on their trial data that they have provided. It looks like bwameth has not been updated in some time to incorporate the updates in the other tools it uses. Hence, had to discard using bwameth. I am using another toolkit BISulfite-seq CUI Toolkit (BISCUIT) for aligning the methyl-seq data. As per the reference for this toolkit, it is appropriate for analyzing bulk and single-cell sodium bisulfite- or enzyme-based DNA methylation/modification data, such as WGBS, capture bisulfite sequencing, RRBS, NOMe-seq, and EM-seq.

Thank you.