| description |
Multiplexed sequencing is widely becoming a popular strategy for increasing the data throughput of sequencing. Given the output of current high-throughput sequencers, it is highly feasible to pool several samples into one sequencing flow cell. Known DNA sequences (barcodes) are incorporated into each library prior to sequencing in order to index and separate the sequenced reads. One strategy for incorporating DNA barcodes is through the use of a mechanism termed template-switching (TS), where the sequence of barcoded TS oligonucleotides is added at the end of first-strand cDNAs. TS is an attractive option given the simplicity of the protocol, which does not require an adaptor mediated step and thus minimizes sample loss. As such, this mechanism has been implemented in several transcriptome analyses such as CAGE, RNA-Seq and small RNA sequencing. Here we report TS artifacts that form due to a process called strand invasion. We describe a strategy that eliminates these artifacts in silico and how such biases can be avoided experimentally. |