removeBimeraDenovo: System Crashes

[khuch123]

I have four samples only and the read number is around 0.6 million but with the step removeBimeraDenovo, system crashes without any error, also the R session does not stop even after pressing the stop button at this command.

I have around 100 Gb of memory and 200 Gb space.

Is it the space issue or something else

[benjjneb]

Can you give us a bit more info? You have way more space/memory than would be needed for the dataset you describe, so it shouldn't be that.

What are the outputs of the following commands:

R.version
packageVersion("dada2")
dim(seqtab)
summary(nchar(getSequences(seqtab)))

Also, can you tell us your sequencing tech, and the exact removeBimeraDenovo command that is crashing?

[khuch123]

Dear Team,

Thank you for responding.
Here are the answers:

R.version
platform x86_64-pc-linux-gnu
arch x86_64
os linux-gnu
system x86_64, linux-gnu
status
major 3
minor 6.1
year 2019
month 07
day 05
svn rev 76782
language R
version.string R version 3.6.1 (2019-07-05)
nickname Action of the Toes

packageVersion("dada2")
1.12.1

dim(seqtab)
Error: object 'seqtab' not found

summary(nchar(getSequences(seqtab)))
Error in is(object, "character") : object 'seqtab' not found

sequencing tech
Illumina HiSeq 2x250 bp paired-end data

exact removeBimeraDenovo command
seqtabNoC <- removeBimeraDenovo(seqtabAll)

Thanks and Regards

[benjjneb]

Can you do dim(seqtabAll) and summary(nchar(getSequences(seqtabAll))) since that is the name of your sequence table.

[khuch123]

Dear Team,
Please find the attached answers

dim(seqtabAll)
[1] 4 51686

summary(nchar(getSequences(seqtabAll)))
Min. 1st Qu. Median Mean 3rd Qu. Max
250 440 447 432 465 483

[benjjneb]

I don't know, that looks pretty normal.
Can you send me an RDS file from this sequence table, i.e. the file from saveRDS(seqtabAll, file="seqtabAll.rds")
I can see if I can reproduce the crash behavior from the same object.

[khuch123]

I have run the command and I could also view this file but how to save it and send it you as no file was generated in my folder.

[benjjneb]

You may need to give a complete file path to save the file where you expect. Otherwise it will save it wherever your working directory is, as shown by getwd().

[khuch123]

Dear Team, Please find the attached file.

…

On Wed, Aug 28, 2019 at 9:06 PM Benjamin Callahan ***@***.***> wrote: You may need to give a complete file path to save the file where you expect. Otherwise it will save it wherever your working directory is, as shown by getwd(). — You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub <#833?email_source=notifications&email_token=AIYQPYFY2SJP5Z6ACKFQ4VTQG2LOTA5CNFSM4IP6JW62YY3PNVWWK3TUL52HS4DFVREXG43VMVBW63LNMVXHJKTDN5WW2ZLOORPWSZGOD5LRIVQ#issuecomment-525800534>, or mute the thread <https://github.com/notifications/unsubscribe-auth/AIYQPYFPF5VN4HKMLVFCV73QG2LOTANCNFSM4IP6JW6Q> .

[benjjneb]

I don't see the file, it probably is too large to attach here.
Can you share it with me via some other way?
My email is benjamin DOT j DOT callahan AT gmail DOT com

[benjjneb]

Thanks for sending the file. I was able to run the following code without incident on my own machine:

library(dada2); packageVersion("dada2") # 1.12.1
fn <- "~/Desktop/seqtabAll.rds"
seqtabAll <- readRDS(fn)
seqtabNoC <- removeBimeraDenovo(seqtabAll, multi=6) # works

Can you confirm that this exact same sequence of commands crashes on your machine?

If so, can you post the full output of sessionInfo() after loading the dada2 R package? So we can see the versions of all the dependency packages as well.

[khuch123]

Thank you
I was able to run it without crash
But now the problem has come upto the command fitGTR as it has been very long that it is running but no error?
Will it eventually end up giving results

[khuch123]

Thank you
I was able to run it without crash
But now the problem has come upto the command fitGTR as it has been very long that it is running but no error?
Will it eventually end up giving results

[benjjneb]

The phangorn GTR tree building does not scale well to thousands or tens of thousands of sequences, and you will probably need to use an external software solution such as RaxML. See further discussion and some suggestions here: #88

[khuch123]

Yes I had seen that discussion.
Thank you
So should I stop that run and rerun with raxml and also could you please post the exact command to be used in place of GTR command as mentioned in the workflow

[benjjneb]

Yes you should probably use raxml.

I don't have exact commands to run, but in that earlier thread there are a couple suggested sets of commands that should get it working.

[khuch123]

Error in raxml(alignment, m = "GTRGAMMAIX", f = "a", p = 1234, x = 2345, :
argument 4 matches multiple formal arguments

[khuch123]

Even after changing the value of x to 5678, the error is still the same