QuantSeq-Pool libraries require paired-end sequencing, as read 2 contains the UMI and sample barcode sequences.
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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: >=50bp. The pipeline will most likely also work for shorter read lengths. However, we should not advise the customer to use <50bp R1.
Read 2: >=22bp. The pipeline will also work for >22bp. However, after extracting the i1 and UMI from the first 22bp of R2, the remainder of R2 will be discarded. Hence, >22bp R2 cannot be used with our pipeline to perform a paired-end QuantSeq-Pool experiment.