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fastqGeneration.md

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Fastq generation

We recommend using bcl-convert from Illumina for fastq generation.

An example samplesheet.csv with typical options is included.

Generate Index Read Fastqs

For ScaleBio Universal libraries the droplet barcode is sequenced in index read I2, while index read I1 is used for multiple libraries (e.g. droplet-instrument lanes). Using bcl-convert this can be achieved by declaring the I2 read as a UMI and generating index read fastqs; using samplesheet.csv settings:

CreateFastqForIndexReads,1
TrimUMI,0
OverrideCycles,Y50;I8;U16;U8Y69

Retain Short Reads

We need to generate a fastq file for (short) barcode reads. In addition ATAC fragments can be quite short after trimming. We hence recommend setting

MinimumTrimmedReadLength,16
MaskShortReads,16

Adapter Trimming

Adapter trimming can be performed directly during fastq generation or (optionally) later on the input fastq files during the workflow. To trim during fastq generation (faster) use

AdapterRead1,CTGTCTCTTATACACATCT
AdapterRead2,CTGTCTCTTATACACATCT

Using pre-generated fastq files as workflow input

Set --fastqDir to the directory containing all fastq files for all samples in an analysis run. The filenames should follow the pattern <Name>_..._<Read>_...fastq.gz, where

  • Name is the library name (libName column in samples.csv)
  • Read is one of R1, R2, I1, I2