Read numbers in Megan "no hits"

User Questions
1

I understand that diamond output read number may be considerably less than diamond input read number, as the output excludes reads for which no alignment is found i.e. excludes “no hit” reads.
How is it then that diamond output reads can end up in the “no hit” node when viewed in Megan?

2

“No Hits” refers to reads that lack any alignments. Typically, this category remains empty when utilizing DIAMOND because DAA files don’t include reads without alignments. However, with BLAST or certain other alignment methods, this category may contain non-empty entries.

If you’re using the --unal option, it controls whether unaligned queries are reported or not.

  • Set it to 0 for “no” if you don’t want unaligned queries to be reported.
  • Set it to 1 for “yes” if you want unaligned queries to be reported.

By default, unaligned queries are reported for the BLAST pairwise, BLAST XML, and SAM formats by DIAMOND.

3

@Anupam
I believe you have described the “not assigned” node, where alignments are present but they don’t exceed preset thresholds. In the “no hits” node, there are no alignments present.

4

Hi @brian

MEGAN applies a threshold for the bitscore of hits, disregarding any hit with a bitscore below this threshold for taxonomy assignment. Consequently, a read may register a hit yet remain unassigned in terms of taxonomy, termed as “not assigned”.

Best,
Anupam

5

@Anupam Yes, that is my understanding too. My question is about the “no hits” node/classification.

6

Hi @brian,

“No Hits” refers to reads that lack any alignments. Typically, this category remains empty when utilizing DIAMOND because DAA files don’t include reads without alignments. However, with BLAST or certain other alignment methods, this category may contain non-empty entries.

Until you’re using the --unal option, it controls whether unaligned queries are reported or not.

  • Set it to 0 for “no” if you don’t want unaligned queries to be reported.
  • Set it to 1 for “yes” if you want unaligned queries to be reported.

By default, unaligned queries are reported for the BLAST pairwise, BLAST XML, and SAM formats in DIAMOND.

On the other hand, “Not Assigned” comprises reads that possess alignments but couldn’t be assigned within the specified classification.

Could you kindly provide us with the DAA file exhibiting this issue along with the DIAMOND command you utilized to generate the DAA file and MEGAN command use MEGANIZE it (logs are more helpful.)? This will help us investigate the matter further.

I have also revised the previous response to enhance clarity.

Best regards,
Anupam