longranger mkfastq : "unrecognised option '--qc' "

Question

[Pierre Lindenbaum]

Hi all,

I'm Pierre Lindenbaum ; I work as a bioinformatician in Nantes/France.

It's the first time I'm invoking longranger mkfastq https://support.10xgenomics.com/genome-exome/software/pipelines/latest/using/mkfastq

this is my command:

module load longranger && \
longranger mkfastq \
        --run="/sandbox/shares/u1087/novaseq/190517_A00797_0007_BHJTYVDMXX" \
        --samplesheet="samplesheet.csv" \
        --qc \
        --output-dir="/sandbox/shares/u1087/lindenb/work/20190522.NOVASEQ" \
        --jobmode=sge

(...)
longranger mkfastq (2.2.2)

(...)

[error] bcl2fastq exited with an error. You may have specified an invalid command-line option. See the full error here:

$ tail /sandbox/users/lindenbaum-p/notebook/2019/20190523.NOVASEQ/HJTYVDMXX/MAKE_FASTQS_CS/MAKE_FASTQS/BCL2FASTQ_WITH_SAMPLESHEET/fork0

<<illumina bcl2fastq help page...>>

Failed to parse the options: unrecognised option '--qc'

How can I fix this error ? I asked the support but I got still no answer. There must be something obvious ?

[Devon Ryan]

What version of bcl2fastq is getting used by longranger mkfastq?

[Pierre Lindenbaum]

BCL to FASTQ file converter
bcl2fastq v2.20.0.422
Copyright (c) 2007-2017 Illumina, Inc.

the ILMN bcl2fastq isn't bundled with longranger ? (I didn't install this software)

[Devon Ryan]

I think you have to provide it, but I've never seen a --qc option. As Genomax said, this is probably a documentation problem.

[Ram]

Isn't the usual reason for "unrecognized option" a difference in versions? Any way you could verify if the version where you got the --qc option and the version loaded by module load are compatible?

[Pierre Lindenbaum]

@ramrs that was my question to devon: I don't know if the ILMN bcltofastq is embedded with longranger

[GenoMax]

No it is not. You need to make it available.

[Pierre Lindenbaum]

ok, but as far as I can see, my longranger is using the latest bcl2fastq 'v2.20.0.422' vs : https://support.illumina.com/content/dam/illumina-support/documents/documentation/software_documentation/bcl2fastq/bcl2fastq2-v2-20-software-guide-15051736-03.pdf

[GenoMax]

That should be fine. That is what I use.

Answer 1

[GenoMax]

There is no --qc option for longranger mkfastq (at aleast with v.2.2.2).

I generally run with something like this:

module load longranger

longranger mkfastq --jobmode=slurm --maxjobs=24 --mempercore=16000 --run=/path_to/FCID  --csv=./SampleSheet_10x.csv --output-dir=/path_to_FCID/mkfq

[Pierre Lindenbaum]

well, the manual says there is this option (?) https://support.10xgenomics.com/genome-exome/software/pipelines/latest/using/mkfastq

--qc    (Optional) Calculate both sequencing and 10x-specific metrics, including per-sample barcode matching rate. Will not be performed unless this flag is specified.

[GenoMax]

Not indicated in in-line help (longranger mkfastq -h).

[Pierre Lindenbaum]

you're right. same here.

[GenoMax]

--qc option is only available for cellranger.

[Pierre Lindenbaum]

well I see it in the longranger mkfastq online doc. I could run without --qc but I want to be sure that there is anything simple that could be fixed :-)

[GenoMax]

You will get regular Illumina format demux report after running longranger.

[ATpoint]

;-P

[Pierre Lindenbaum]

:-D

sorry , I'm new here.

I'm away from the lab. I'll test without --qc tomorrow.

[GenoMax]

Use a simple samplesheet like this with command I posted in answer.

Lane,Sample,Index
*,Sample1,SI-GA-A12
*,Sample2,SI-GA-C7
*,Sample3,SI-GA-C8

You will get folders for each sample at the end of the run.

If the same pool did not run in all lanes then you will add a lane number instead of *.

[Devon Ryan]

Does * actually work to specify "all lanes"? I just remove the Lane column completely in such cases.

[GenoMax]

Yes * works.