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bcl2fastq.md

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convert bcl files to fastq files

bcl2fastq is a software developed to convert bcl files to fastq files.

Here we use bcl2fastq v2.20.

Official website: https://emea.support.illumina.com/sequencing/sequencing_software/bcl2fastq-conversion-software.html

A brief tutorial: https://www.10xgenomics.com/cn/support/software/cell-ranger/latest/analysis/inputs/cr-direct-demultiplexing-bcl2fastq

1. Information of index adapters

A practical guide to single-cell RNA-sequencing: https://genomemedicine.biomedcentral.com/articles/10.1186/s13073-017-0467-4

In this study, we used Novaseq 6000 platform for sequencing, here's a brief introduction to the platform including the description of files in the output folders: https://support.illumina.com/content/dam/illumina-support/documents/documentation/system_documentation/novaseq/1000000019358_17_novaseq-6000-system-guide.pdf

In the sequencing output dir, you can see a file named RunInfo.xml, which contains lists the run name, number of cycles in each read, whether the read is an Index Read, and the number of swaths and tiles on the flow cell.

Information for the reads:

<Reads>
	<Read Number="1" NumCycles="101" IsIndexedRead="N" IsReverseComplement="N"/>
	<Read Number="2" NumCycles="10" IsIndexedRead="Y" IsReverseComplement="N"/>
	<Read Number="3" NumCycles="10" IsIndexedRead="Y" IsReverseComplement="Y"/>

Then we can use --use-bases-mask=Y101,I10,I10, and for that the IsIndexRead="Y" and IsReverseComplement="Y" for read number 3, we need to use ==the sequences of index1 and reversed complemented sequences of index2== as the input index sequences for bcl2fastq.

2. SampleSheet.csv

The main aim of using samplt sheet is to demultiplex reads the lane into different samples by the index sequences. So we can reserve only the index information as following:

Note

Remember that the header is [Data]

[Data]
Sample_ID,Index,Index2
cell1,GACCAAGTTA,GATTCACGAC
cell2,CTCCGCTTAT,GATTCACGAC
cell3,ATTAAGGTCG,GATTCACGAC

3. run the software

bcl2fastq --use-bases-mask=Y101,I10,I10 \
--create-fastq-for-index-reads \
--minimum-trimmed-read-length=8 \
--mask-short-adapter-reads=8 \
--ignore-missing-positions \
--ignore-missing-controls \
--ignore-missing-filter \
--ignore-missing-bcls \
-r 3 -w 3 -p 10\
-R ${FLOWCELL_DIR} \
--output-dir=${OUTPUT_DIR} \
--sample-sheet=${SAMPLE_SHEET_PATH}

Note

When failed with error reporting Too many open files, check this document:https://www.howtogeek.com/805629/too-many-open-files-linux/