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hildebra / lotus2

Amplicon sequencing pipelines suitable for SSU (16S, 18S), LSU (23S, 28S) and ITS.
http://lotus2.earlham.ac.uk/
GNU General Public License v3.0
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sdm dereplicated output file empty #78

Open TJVUMC opened 2 months ago

TJVUMC commented 2 months ago

Hi, I tried running lotus2 but got the following error: This is sdm (simple demultiplexer) 2.17 beta.

Checking for switched pairs. Run with 12 cores.At lotus2/SRR13436190_1.fastq:

Undecided fastq version.. sdm 2.17 beta Input File: /home/sarmat2//lotus2/SRR13436190_1.fastq,lotus2/SRR13436190_2.fastq Output File: output//tmpFiles//demulti.1.fna

Reads processed: 79,819; 79,629 (pair 1;pair 2) Rejected: 79,810; 79,758 Accepted (High qual): 0; 0 (0; 0 end-trimmed) Accepted (Mid qual): 0;0 Singletons among these: 0; 0 Bad Reads recovered with dereplication: 0 Min/Avg/Max stats Pair 1

hildebra commented 2 months ago

Hey, can you please switch to sdm 2.18? This might already solve the problem, it looks like the quality of the reads was either wrongly read in or that the quality of the reads is overall too bad (seems unlikely). Which sequencing platform was used, AVITI by any chance? best, Falk

TJVUMC commented 2 months ago

Hi Falk, Thank you for looking up. The V4 hypervariable region of bacterial 16S rRNA gene amplified with 515 forward and 806 reverse primers using a MiSeq Illumina platform (150‐bp read length, paired‐end protocol). By switching to sdm 2.18, you mean updating lotus2 too? Please suggests. Thanks

Sincerely, Tulsi

From: Falk Hildebrand @.> Sent: Friday, August 16, 2024 1:14 AM To: hildebra/lotus2 @.> Cc: Joishy, Tulsi K @.>; Author @.> Subject: Re: [hildebra/lotus2] sdm dereplicated output file empty (Issue #78)

Hey, can you please switch to sdm 2.18? This might already solve the problem, it looks like the quality of the reads was either wrongly read in or that the quality of the reads is overall too bad (seems unlikely). Which sequencing platform was used, AVITI by any chance? best, Falk

- Reply to this email directly, view it on GitHubhttps://github.com/hildebra/lotus2/issues/78#issuecomment-2292884916, or unsubscribehttps://github.com/notifications/unsubscribe-auth/BG6DQLEWC6RBJ3OLESBG2ITZRWKB7AVCNFSM6AAAAABMS47HBCVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZDEOJSHA4DIOJRGY. You are receiving this because you authored the thread.Message ID: @.***> [ WARNING : This email came from an external source. Please treat this message with additional caution.]

hildebra commented 2 months ago

Hey Tulso, yes exactly, switching to the latest lotus2 version (only available on github currently). However, alternatively you could update to sdm2.18 via conda (this is already on conda), eg run "mima install sdm=2.18"

TJVUMC commented 2 months ago

Hi, I just updated the lotus2 and sdm, but I am not getting even Lotusprogout.log in output. Did I screwed up whole pipeline? Please suggest. Thank you. Tulsi

From: Falk Hildebrand @.> Sent: Friday, August 16, 2024 10:10 AM To: hildebra/lotus2 @.> Cc: Joishy, Tulsi K @.>; Author @.> Subject: Re: [hildebra/lotus2] sdm dereplicated output file empty (Issue #78)

Hey Tulso, yes exactly, switching to the latest lotus2 version (only available on github currently). However, alternatively you could update to sdm2.18 via conda (this is already on conda), eg run "mima install sdm=2.18"

- Reply to this email directly, view it on GitHubhttps://github.com/hildebra/lotus2/issues/78#issuecomment-2293687711, or unsubscribehttps://github.com/notifications/unsubscribe-auth/BG6DQLFREYWZIXWHJ6QZ5F3ZRYI4VAVCNFSM6AAAAABMS47HBCVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZDEOJTGY4DONZRGE. You are receiving this because you authored the thread.Message ID: @.***> [ WARNING : This email came from an external source. Please treat this message with additional caution.]

hildebra commented 2 months ago

Hey, try to run the examples.. (in README) if that doesn't work, rm lotus2 dir and reinstall via github.. best, Falk

TJVUMC commented 2 months ago

Okay! Thank you.

Tulsi

Sent from Outlook for Androidhttps://aka.ms/AAb9ysg

From: Falk Hildebrand @.> Sent: Saturday, August 17, 2024 5:34:43 AM To: hildebra/lotus2 @.> Cc: Joishy, Tulsi K @.>; Author @.> Subject: Re: [hildebra/lotus2] sdm dereplicated output file empty (Issue #78)

Hey, try to run the examples.. (in README) if that doesn't work, rm lotus2 dir and reinstall via github.. best, Falk

— Reply to this email directly, view it on GitHubhttps://github.com/hildebra/lotus2/issues/78#issuecomment-2294821395, or unsubscribehttps://github.com/notifications/unsubscribe-auth/BG6DQLHOFEIPCRDWGSHVJHLZR4RMHAVCNFSM6AAAAABMS47HBCVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZDEOJUHAZDCMZZGU. You are receiving this because you authored the thread.Message ID: @.***>

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paulzierep commented 3 weeks ago

When looking into the same issue on the Galaxy server, I found that the default value of lotus for2 -derepMin is 8:1,4:2,3:3 (so if you have only one samples your read needs to be repeated 8 times, if I got that correct ?); this could well lead to all reads being rejected. Would it maybe make sense to make the default value more moderate ?

hildebra commented 3 weeks ago

Hey, yes the current derepMin is set up to require each (unique) read to be present 8 times in a single sample, or 4 times across two different samples (or 3 times across 3 samples) . These options were create to match well for gut microbiomes amplicons sequenced at ~15,000 reads/sample. E.g. in soils this might be too strict (they're too diverse), or if the sequencing depth is substantially lower. The process is admittedly a bit arbitrary, but works in our experience really good at removing false positives. The best solution would really be to have an automatic estimation based on sequencing depth & diversity, but this would be its own little project. I'll look if I can add some defaults based on input size. best, Falk