What read length should I use for QuantSeq-Pool Reads 1 and 2?

QuantSeq-Pool libraries require paired-end sequencing, as read 2 contains the UMI and i1 sample barcode sequences.

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.

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 lengths are not recommended as this will capture the poly(T) stretch. Reading through homopolymer stretches can negatively impact read quality, and will require more complicated trimming.

The QuantSeq-Pool 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.