fastqGeneration.md

Fastq generation

The workflow can be started from a sequencer runFolder (bcl) [--runFolder]. In that case fastq files will be generated internally, using Illumina bcl-convert.

Alternatively it is also possible to generate fastq files upstream, for example when the ScaleCROP library is multiplexed together with other libraries during sequencing at a core facility or with a ScaleRNA library. In this case we recommend using bcl-convert.

An example samplesheet.csv with typical options is included. Here all 96 i5 barcode sequences from the PCR plate are merged into one set of fastq files. If an index1 (i7) read is used to demultiplex the ScaleBio CRISPR library with other libraries in the sequencing run, usage of the index column should contain the constant i7 sequence of the CRISPR library, which can be found here in forward orientation: 3lvlCRISPR_p7.txt. This becomes especially relevant when working with multiple final distribution plates. In this case, each sample name for each row in the samples.csv needs to corresopnd to each sample found PER PLATE uniquely.

Index reads

For ScaleBio RNA and CRISPR libraries the RT and ligation barcodes are included in read 1, the PCR plate barcode is in the index1 read, while the PCR well barcode is in the index2 read. Hence we need to tell bcl-convert to generate index read fastqs using the samplesheet.csv setting:
CreateFastqForIndexReads,1

Using pre-generated fastq files as workflow input

Set --fastqDir to the directory containing the fastq files. The file names should follow the pattern <LibraryName>_..._<Read>_...fastq.gz, where

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