Technical note : Molecular Index counting adjustment methods

BD Precise assays are fast, high-throughput, next-generation sequencing (NGS) library preparation kits tailored for small quantity RNA samples, such as single cells, using patented BDTM Molecular Indexing (MI) technology with Sample Index (SI) to label individual mRNA transcripts. During reverse transcription, the BD Precise assays apply a non-depleting pool of 6,561 MI barcodes (or 65,536 barcodes for BD Precise whole transcriptome amplification assay) for stochastic and unique labeling of otherwise identical mRNA transcripts to generate unique cDNA.1 In addition to MI, 96 SI barcodes are employed to identify the sample origin of each transcript according to the well position in the 96-well BD Precise plate. These unique barcoded primers are then used to label polyadenylated RNA transcripts in each of the 96 wells, followed by a pooling step into a single tube for a multiplex PCR target enrichment and sequencing adapter attachment (BD Precise targeted assay). The resulting library is then ready for NGS using compatible Illumina sequencers and sequencing kits. Each of the sequencing reads are then processed to identify the MI, SI and target gene using a BD primary analysis pipeline (Figure 1).