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.
The optimal length for read 1 is 75 - 100 bp. However, longer read lengths can also be used. 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, read trimming to remove poly(A) homopolymers, adapter sequences and low quality bases will ensure you retain maximal insert read lengths for mapping purposes.
The QuantSeq-Pool pipeline available on the Lexogen Tools Github Page is compatible with the following read length parameters:
Read 1:
Read 2: