QuantSeq-Pool libraries require paired-end sequencing, as read 2 contains the UMI and sample barcode sequences.
We recommend a length of 22 bp for read 2 in order to fully read-out the UMI and sample barcodes. 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 optimal length for read 1 is 75 - 100 bp. However, longer read lengths can also be used. Per The optimal length for read 1 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 the more inserts will be fully covered and start to read into the poly(A) stretch and adapter sequences. However
Regardless of read length, read trimming trimming 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 in order to fully read-out the UMI and sample barcodes. 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 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 not recommendedcannot be used for mapping.