q2-feature-classifier query

Hello sir,

I have been confused about how to set the p-trunc-len, --p-min-length, --p-max-length value, we have performed Illumina sequencing (MIseq) at Biokart, Bangalore for our bacterial sample. My sequence maximum length was 300 bp and minimum length was 35 bp and our primer sequence was primer

I didn't trim or truncate my sequence in deionising step, I gave zero value for that option (--p-trim-left
--p-trunc-len) . I am so scared right now whether setting value will cause us to lose significant amplicon

Could I set zero value for all options or --p-trunc-len 0 -p-min-length 35 --p-max-length 300 ?

qiime feature-classifier extract-reads
--i-sequences 85_otus.qza
--p-f-primer GTGCCAGCMGCCGCGGTAA
--p-r-primer GGACTACHVGGGTWTCTAAT
--p-trunc-len 0
--p-min-length 0
--p-max-length 0
--o-reads ref-seqs.qza

Kindly help me sir.

you can always look at the number of input/output sequences to count how many reference sequences are lost.

that’s fine, as long as the primers are not in the reads.

sounds reasonable.

Much of this decision-making depends on your data, the sequencing protocols used, etc, so I can only provide approximate suggestions! But usually the read extraction step is not a very risky one.

Good luck!

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Thank you so much for your reply and suggestions sir. I will try as you mentioned.

Hello sir,

As per your suggestions, I ran qiime feature-classifier extract-reads command-line. My NCBI_REF sequence got reduced from 21,624 to 16 sequences after the extraction.

This was the command line I ran

I ran this command-line multiple times, by changing value trunc length --p min and --p max length, but my sequence count got reduced even more bad. It came three and five sequence finally.

What should I do further?

the primer sequences are not being found in the reference sequences, or your trimming settings are wrong. Exactly why I cannot say (no hits? reference sequences are already trimmed?) but I recommend manually inspecting some sequences to determine why.

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Thank you so much for your reply sir. I will look in to it.

Hello sir,

I got one doubt whether I have to give reverse complement of reverse primer or reverse primer in q2-feature-classifier extract reads commandline.

Reverse complement of reverse primer : GGACTACDBGGGTWTCTAAT

or

Reverse primer: ATTAGAWACCCVHGTAGTCC

Thanking you in advance for your help.

Please see the help docs for specific usage details specific to the version that you are running:
qiime feature-classifier extract-reads --help

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Thank you so much for your reply sir.

My problem got solved, reverse compliment of reverse-primer works good. I didn’t loose more reads. 98% of reads got retained.

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