Guide me to generate .fna and .biom file

[Subhrangshu Mandal]

Dear PICRUSt2 Developer,

I hope this message finds you well. I am currently encountering an issue while trying to generate .fna and .biom files from QIIME 2. I have single-end reads from amplicon sequencing performed on the Ion Torrent platform, and I am facing difficulty with the conversion process.

As a beginner in this field, I have attempted the task several times, but I am still unable to successfully generate the files. I would greatly appreciate it if you could kindly provide me with guidance or a terminal copy of the steps to generate these files.

Thank you for your time and assistance. I look forward to your support.

Best regards,

[Robyn Wright]

Hi there,

I don't personally have experience with Ion Torrent amplicon sequencing files, but what format do you have them in? Are they demultiplexed fastq files (i.e. a single fastq file for each sequence)? If yes, you should be able to use QIIME2 for processing still. My lab has tutorials for the first part of the processing here, but you may need to modify the importing steps (you can see some other options here). I'm also not sure how the denoising steps will be for Ion Torrent data, but you could use OTU clustering instead if you run into problems. 

Robyn

[Subhrangshu Mandal]

In Ion Torrent files are single-end read no need demultiplex the data . thank you for tutorial I am walking through the tutorial , didnt found the .fna file creation strategy

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[Robyn Wright]

The .fna file creation will be right at the end in step 10. Note that the file just needs to fit the fasta format (i.e. two lines per sequence, the first one starting with > and containing a unique sequence name, and the second containing the DNA sequence), and the extension doesn't actually need to be .fna. Following the tutorial, it will be in the deblur_output_exported folder at the end.

Robyn

[Subhrangshu Mandal]

i tried but found this issue

(picrust2) sudiptobiolab@sudiptobiolab-Latitude-5420:~/Qiime2_trialset/Test1151$ picrust2_pipeline.py -s dna-sequences.fasta \
-i feature-table.biom \
-o picrust2_out \
--processes 8 --stratified --per_sequence_contrib
No FASTA file found in specified directory. Expected to find one of:
/home/sudiptobiolab/anaconda3/envs/picrust2/lib/python3.8/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.fna.gz
/home/sudiptobiolab/anaconda3/envs/picrust2/lib/python3.8/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.fasta.gz
/home/sudiptobiolab/anaconda3/envs/picrust2/lib/python3.8/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.fna
/home/sudiptobiolab/anaconda3/envs/picrust2/lib/python3.8/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.fasta


In addition to the missing FASTA file, the following expected file(s) could not be found:
/home/sudiptobiolab/anaconda3/envs/picrust2/lib/python3.8/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.tre
/home/sudiptobiolab/anaconda3/envs/picrust2/lib/python3.8/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.hmm
/home/sudiptobiolab/anaconda3/envs/picrust2/lib/python3.8/site-packages/picrust2/default_files/prokaryotic/pro_ref/pro_ref.model

help me out

running 2.5.3 version

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[Robyn Wright]

Hi there,

That error is suggesting that the expected files aren't in the default location. Can you check whether the files are actually at that location? /home/sudiptobiolab/anaconda3/envs/picrust2/lib/python3.8/site-packages/picrust2/default_files/prokaryotic/pro_ref/

If they aren't there, you could try re-installing PICRUSt2, or you could just copy them across to that location from here: https://github.com/picrust/picrust2/tree/master/picrust2/default_files/prokaryotic

Robyn

[Subhrangshu Mandal]

I tried and it predicted but i lost the taxon/genus name from the table please look after this file

sequence	K00001	K00002	K00003	K00004	K00005	K00006	K00007	K00008	K00009	K00010	K00011	K00012	K00013	K00014
02d7ef078e65aeeb001dd17588fc7bdd	1	0	1	1	2	0	0	0	0	0	0	2	1	1
0301878e8a67a9a5324e646459c5cd25	0	0	1	0	1	0	0	2	0	0	0	0	1	1
035b05891bd1068e393c06ad946c5e8e	2	0	1	1	0	0	0	0	0	0	0	2	1	1
036cdb0da476c4c203caba7a642cdff6	1	0	1	1	0	0	0	0	0	1	0	1	1	3
04f1d0f85b3a3b7bb7fc423af172f51b	4	0	1	0	0	0	0	0	0	3	0	1	1	2
053b04d33d307d47c406fc713358ebca	0	0	1	0	0	0	0	0	0	0	0	2	1	1
071286f0c12cc837e21dad95cc459f77	1	0	1	1	0	0	0	2	0	1	0	1	1	1
0fb7d1f2b36169af66c79a57c9564120	0	0	3	1	0	0	0	0	0	0	0	2	1	1
0ff86a7171f5c719df4585a7fa584329	1	0	1	0	0	0	0	0	0	0	0	1	1	1
1042df6ad680b3dcbfba75a4dbedf07b	0	0	1	0	1	0	0	0	0	0	0	1	1	1
118980664be12644fe7b4b682cfb4cdb	0	0	1	0	0	0	0	2	1	1	0	2	1	1
12272ae04c6aaf3612a4939956b1a387	1	0	2	0	0	0	0	0	1	0	0	0	1	1
122e4818c1ad5124e61accc5da0c0a64	0	0	1	1	0	0	0	0	0	0	0	1	1	1
13180d572c500599a6805cc3637a183f	0	0	1	0	4	0	0	1	2	0	0	2	1	2
177edca42928ca1d0b02007fc0e45eaf	1	0	1	1	0	0	0	0	0	0	0	1	1	1
1783d6306d68509799905dd45fdc4e80	2	0	3	1	0	0	0	0	0	1	0	2	1	1
1830f3d2533e23ab9c0a3db602cb8bb2	2	0	1	1	0	0	0	0	0	0	0	1	1	2
1bd3a5532c491c13504879fa1f4ce39b	1	0	0	0	2	0	1	0	1	3	0	1	1	1
1c6d2f924127e235a24eb4eaf3ddfa8d	0	0	1	0	0	0	0	0	0	0	0	1	1	1
1eee86e1cb4183746c6c2e182f8d96cd	2	0	3	1	0	0	0	0	0	1	0	2	1	1
22df54ec863835af209ffb3bc06840f4	0	0	0	0	1	0	0	0	1	0	0	1	1	1
2628ed6c99dea2eb05f436f6e3d8a09f	5	0	1	0	0	0	0	1	0	0	0	2	1	1
267a0a6a32499eae9143e78fa9e6bd5e	0	0	1	0	0	0	0	0	0	0	0	1	1	1
293de075876ebbb973439b1f77a22bba	3	0	1	1	0	0	0	1	0	0	0	4	1	1
2baa7abc21b2fe754271932db496af91	2	0	1	1	0	0	0	0	0	0	0	1	1	1
2bda7e58c0011e1286145c3500c5900e	1	0	2	0	0	0	0	0	0	2	0	1	3	1
2deeae44a2364de61090f229ee4bc387	0	0	3	1	0	0	0	0	0	0	0	2	1	1
304e7a0623732a548ca2f13e887c3225	0	0	1	0	0	0	0	0	0	0	0	1	1	1
30c103d145d411e51b451d0096b8c5c8	0	0	0	0	1	0	0	0	1	0	0	1	1	2
385c415dec8c07d14b6ad398f8a08609	2	0	1	0	0	0	0	1	0	1	0	1	1	2
3a9bf833c9e4918b069d4d6bc7e6d0cb	1	0	3	1	0	0	0	0	0	0	0	2	1	1
3b548961207768d6ece2b1a90dc400f8	0	0	0	0	0	0	0	0	0	0	0	1	1	1

its like this (a short portion of .tsv file)

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[Robyn Wright]

This table looks as I would have expected - I'm a bit confused about what you think is missing?

Robyn

[Subhrangshu Mandal]

the sequence identification like genus name where sequence  is placed

bacteria genus name insted of this : 1830f3d2533e23ab9c0a3db602cb8bb2

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[Robyn Wright]

Usually the ASV classifications are stored in a separate file (if you ran QIIME2 then usually you also export the taxonomy information separately) and would only be in your PICRUSt2 output if they were a part of the sequence name (with no spaces) in your feature table and sequence file. 

Robyn

[Subhrangshu Mandal]

while running picrust2 i got this message

Running PICRUSt2 for SM8...

Warning - 51 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: 9b3eac8acdd9348c9df3e9c19e0d15d5, 3400a1ff6773f9ce8a3eddec415d5d98, a628e6ef21d77075bb3ddd15c9152653, d8da0eaa936907e6af65b212c3a4ce83, fd0ddeb660c00120cc330d8d08478eb6, 75a9ddee945ec32891b16e9e1bd0e6da, 454131b0e068cc016bf85437480a013f, 1d9a8578e8fb365de4f65af85e7980ba, 59cd12979235d1da987258075d4070a4, e733d1644146838c04b05e08786f17be, 25d6b3b7a4abaf65be5168456aa5951f, b5a7d3de2610054755babee1bbcdac19, f969849ed5771f474dfebda73c2578c4, 7d76e4dfc784a3d4ff42cacced93e9c1, 19838ecb9e274e6e85516c302a0ea36d, 575646ab1d7419c5ccf60b228404bb66, b46ae1770ac405642720153fc406538a, 450b7a61c40e2c5a95764fb4414f1d10, 546ad46f40e392afbf8b8ba21ea3078e, d0839d29d49f6d3ad93f920b886d1a7f, 9aabb2a4ec54757ef021a4e46efa63a7, 79d84e6c295f1df1537b6e805022b85c, e4ad9a1f763dadd1c710277b2520c8e4, 54cf40a828cb95f7324b919462935c9a, d3e821b367e1368dd43519d434f3652f, d3470800db7989ab7b011a9b64a54334, a72b606b413da6d92e396e5c29b04ec1, 283f1632e2061541cb12561305ac894d, f355a037c2e8d934998989ae276cd107, b3e3f8e41dc448facc002082b4f0c8a3, cf25d97085672811cfbaac3a84396708, cd45daebc574b56545e936b6cd69b356, 2d80a38ee84b2a0cf73b4f318b6953ee, 5bfafd4f1fca274e94f756e11fbf816c, 27acc55345e6803942ff6842c1697e46, c7250785474f340413656fe146935538, c410f9a6e4c1d5d1fab0c652ff986acc, a9b3bcb19d8b6937b977e14dd0ab8f04, 1c00b498d186fb87f00224024aa85574, 4c8f3468b114701645fa122ab07b23c4, c7998ed379bfac4b5504a7813dd00343, 7110b91f042acbe373d0251776ffd2f5, 575253286245debb38f4fd6fd648148b, a424406678ff1495c969a476cd7f91e9, d98864a5edfb0759886bd1c8871de08f, c2d66a8cde36859b342230948a5e399d, 78e430e0cf1c480069e5f9a660f9d770, dc76557ed2efe26459fec06ad890f67c, 6d0a121ca114fd70d44b2f93e9a6a070, 70c24120c42a1ef67d256f0845dd3aff, 736f41f7d7d0842cf7ff3312a4bc04bc

3 of 352 ASVs were above the max NSTI cut-off of 2.0 and were removed from the downstream analyses.

3 of 352 ASVs were above the max NSTI cut-off of 2.0 and were removed from the downstream analyses.

Finished PICRUSt2 for SM8

so these sequence ids are removed from the KO_predict.tsv  while merging asv with ko thease are not sitting in appropriate row

how can i mitigate this ?

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[Robyn Wright]

Hi there,

Unfortunately there isn't really any way around this. This means that there was nothing in the reference database that was a good match to these sequences, so we can't make predictions for them. You could see this section of the FAQ - it talks about when no ASVs match, but could be relevant depending on how you've made your sequences file. Do you know what these sequences are classified as, or could you run them through BLAST to see what the closest matches are like? 

Best wishes,

Robyn