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Can I use single-read sequencing for QuantSeq-Pool libraries?

No. QuantSeq-Pool requires asymmetric, paired-end sequencing. It is necessary to sequence Read 2 for sample identification, i1 barcode demultiplexing and UMI deduplication.

Read 1 is generated towards the poly(A) tail and directly corresponds to the mRNA sequence. This read will be trimmed and mapped to report transcript expression levels.

Read 2 will contain the Unique Molecular Identifier (UMI) followed by the i1 sample-barcode. This read is required for demultiplexing and read de-duplication following alignment.

See: What read length should I use for QuantSeq-Pool Reads 1 and 2? for recommended read lengths.

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