Analysis of q2-picrust2 pipeline based generated qza files

[Rashmi_Ira]

Hye @gmdouglas, @jairideout and @Mehrbod_Estaki,

Please look into the follow-up question

https://forum.qiime2.org/t/picrust2-removed-3-751-input-sequences-aligned-poorly-to-reference-sequences/26892/5?u=rashmi_ira

@Rashmi_Ira

Dear @gregcaporaso,

I am also having a same doubt.
Me too performing this analysis for a very first time, trying to use q2-picrust2 with my environmental samples (sludge samples).
Here is command I have used:

qiime picrust2 full-pipeline \

--i-table merged-table_EF_RW.qza
--i-seq merged-rep-seqs_EF_RW.qza
--output-dir q2-picrust2/q2-picrust2_output
--p-placement-tool epa-ng
--p-threads 32
--p-hsp-method mp
--p-max-nsti 2
--verbose

In response of the above command I was getting this warning.

Warning - 2 input sequences aligned poorly to reference sequences (--min_align option specified a minimum proportion of 0.8 aligning to reference sequences). These input sequences will not be placed and will be excluded from downstream steps.

Thanks in advance!

Best Regards,
Rashmi

[Rashmi_Ira]

Dear @gregcaporaso,

I am also having a same doubt.
Me too performing this analysis for a very first time, trying to use q2-picrust2 with my environmental samples (sludge samples).
Here is command I have used:

qiime picrust2 full-pipeline \

--i-table merged-table_EF_RW.qza
--i-seq merged-rep-seqs_EF_RW.qza
--output-dir q2-picrust2/q2-picrust2_output
--p-placement-tool epa-ng
--p-threads 32
--p-hsp-method mp
--p-max-nsti 2
--verbose

In response of the above command I was getting this warning.

Warning - 2 input sequences aligned poorly to reference sequences (--min_align option specified a minimum proportion of 0.8 aligning to reference sequences). These input sequences will not be placed and will be excluded from downstream steps.

**

This is the set of poorly aligned input sequences to be excluded: 8fad6efc85152fc02447ff4a180b8ad9, 02b86a50afd72eee48f6669b5aa67fa9

**
What it means????
Are my results good??

**43 of 3158 ASVs were above the max NSTI cut-off of 2.0 and were removed from the downstream analyses.

**43 of 3158 ASVs were above the max NSTI cut-off of 2.0 and were removed from the downstream analyses.

Please suggest whether, these values of positive interest or not??? If not, then how can I play with parameter **

--p-max-nsti 2

**

Any suggestion or comment will be appreciated.

Best Regards,
Rashmi

[gmdouglas (Gavin Douglas)]

Hi @Rashmi_Ira,

I no longer maintain PICRUSt2 and am no longer following the activity on this and other forums. For future PICRUSt2 questions you could also reach out to the current maintainers on the picrust-users google group: https://groups.google.com/g/picrust-users?pli=1

However, in this case I would BLAST some of the sequences that failed to see if they were non-bacterial or otherwise poorly characterized lineages (or perhaps a chimeric sequence). These would be reasons why the sequences would not be alignable.

You can also check out this FAQ answer: Frequently Asked Questions · picrust/picrust2 Wiki · GitHub

Cheers,

Gavin

[Rashmi_Ira]

Thank you @gmdouglas for your response.