A dual indexed approach to small RNA sequencing library preparation for PRO-seq
AUTHORS
ABSTRACT
Advances in next generation sequencing (NGS) technologies have vastly increased the number of samples that can be
sequenced together and reduced the cost per sample. To fully utilize the increased capacity of standard sequencers, samples
must be multiplexed. Sample multiplexing allows libraries to be pooled and sequenced together on a single sequencing run.
Multiplexing requires libraries that have been prepared with a unique 6-12 bp sequence (index). The most efficient and costeffective sequencing platforms rely on paired end sequencing methodologies to improve multiplexing efficiency. While libraries
with a single index can be sequenced on paired end sequencers they reduce the efficiency of demultiplexing and increase the
sequencing cost due to index mis-assignment. However, some specialized library preparation protocols, like the TruSeq Small
RNA protocol, that rely on custom sequencing adapters have not been updated by Illumnia to include a second index.
Commercially available kits for creating small RNA paired end libraries are expensive and do not provide the level of
transparency required to adapt the kits to custom protocols. Here, we provide a dual-indexed approach to small RNA library
preparatio that reduces the cost per library by nearly half (compared to the NEXTFLEX v3 kit) and includes all sequences for
the oligonucleotides required. While this workflow was developed for PRO-seq libraries, deviations from the original TruSeq
Small RNA library preparation protocol are denoted and alternate reagents are provided to facilitate adaptation of existing
protocols.