QuantSeq-Pool libraries require paired-end sequencing, as read 2 contains the UMI and i1 sample barcode sequences.
The optimal length for read 1 Read 1: Optimal read length is 75 - 100 bp.
Read lengths <50 bp are not recommended as shorter reads are more likely to be lost through trimming, resulting in lower alignment rates.
Read lengths >100bp can also be used, It is important to note that per base sequencing quality will typically decrease as read-length increases, as more inserts will be fully covered and start to read into the poly(A) stretch and adapter sequences.
Regardless of read length, trimming of Read 1 should be performed as a first step in data analysis, to remove poly(A) homopolymers, adapter sequences and low quality bases. This will ensure you retain maximal insert read lengths for mapping purposes.
We recommend a length of 22 bp for read 2 Read 2: Recommended read length is 22 bp, in order to fully read-out the UMI and sample barcodes.
Shorter reads are not recommended as you will not be able to identify reads belonging to each sample.
Longer read 2 read lengths are not recommended as this will capture the poly(T) stretch. Reading through homopolymer stretches can negatively impact read 2 quality, and will require more complicated trimming.
The QuantSeq-Pool pipeline Analysis Pipeline available on the Lexogen Tools Github Page is compatible with the following read length parameters:
Read 1: >=50bp. The pipeline will most likely also work for shorter read lengths. However, we do not recommend using read lengths <50 bp for QuantSeq-Pool.
Read 2: >=22bp. The pipeline will also work for reads >22bp. However, after extracting the i1 and UMI from the first 22bp of R2, the remainder of R2 will be discarded and are therefore cannot be used for mapping.
...