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NextSeq 2000 bcl2fastq conversion

Dear all,

I have a small RNA sequencing run (QIAseq miRNA low input) in NextSeq 2000 sequencing machine. I have all the related files but when I ran bcl2fastq --barcode-mismatches 0 in the directory where all the files are, I do get the following error:

ERROR: bcl2fastq::common::Exception: 2022-Mar-07 22:18:49: No such file or directory (2): /sw/apps/bioinfo/bcl2fastq/2.17.1/src/bcl2fastq/src/cxx/lib/data/BclFile.cpp(103): Throw in function static void bcl2fastq::data::BclFile::getAndVerifyFileName(const boost::filesystem::path&, bcl2fastq::common::LaneNumber, bcl2fastq::common::TileNumber, bcl2fastq::common::CycleNumber, bool, boost::filesystem::path&)
Dynamic exception type: boost::exception_detail::clone_impl<bcl2fastq::common::IoError>
std::exception::what: Unable to find BCL file for 's_1_1101' in: /crex/proj/snic2020-16-229/Toxoplasma_bcl_delivery05646/INBOX/P23001/220202_VH00203_129_AAANKJFM5/Data/Intensities/BaseCalls/L001/C1.1

Based on the post here it seems that NextSeq 2000 uses CBCL instead of BCL. Is there any specific option for bcl2fastq to specify the method or similar thing to circumvent the error?

Thank you.

sequencing bcl2fastq fastq
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updated by rds • rds • written by Apex92 • Apex92

1 answer

bcl2fastq does not support NextSeq 2000, only NextSeq 500, as written in the release notes for bcl2fastqs 2.20

bcl2fastq version 2.20.0 is used to convert bcl files to FASTQ, and is compatible with MiniSeq, MiSeq, NextSeq 500, all HiSeq (2000, 2500, 3000, 4000, and HiSeqX), and NovaSeq 5000/6000 systems running RTA version 1.18.54 and above

Try bclconvert

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written by Carambakaracho Carambakaracho

Thank you for you comment - could you also please share a quick command of the bclconvert?

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written by Apex92 • Apex92

Not off hand, a colleague used it once for a similar task when he failed to convert Nextseq 2000 bcls. I recall, the usage is very close to bcl2fastq

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written by Carambakaracho Carambakaracho

Carambakaracho is correct. NS 2K requires bcl-convert for data processing. Syntax is very similar to bcl2fastq.

bcl-convert --bcl-input-directory FCID_dir --output-directory Unaligned_data --sample-sheet ./SampleSheet.csv --bcl-num-conversion-threads 6 --bcl-num-compression-threads 6 --bcl-num-decompression-threads 6

Be careful with number of threads. Since you have NS 2K data your bcl files should be compressed so last two options can be used.

You can run bcl-convert -h to access in-line help.

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written by GenoMax GenoMax

In my experience, bcl2fastq works totally fine for NextSeq 2000 data! This error may have been due to a missing/corrupted file or some other technical issue.

NextSeq 2000 isn't listed in these bcl2fastq release notes because they were published in 2017, before the NextSeq 2000 was released in 2020 (the same applies to the 2019 release notes). Regardless, NovaSeq runs also output CBCL data, so the CBCL format isn't the issue.

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written by rds • rds

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