A highly multiplexed and sensitive RNA-seq protocol for simultaneous analysis of host and pathogen transcriptomes

The ability to simultaneously characterize the bacterial and host expression programs during infection would facilitate a comprehensive understanding of pathogen-host interactions. Although RNA sequencing (RNA-seq) has greatly advanced our ability to study the transcriptomes of prokaryotes and eukaryotes separately, limitations in existing protocols for the generation and analysis of RNA-seq data have hindered simultaneous profiling of host and bacterial pathogen transcripts from the same sample.

Here, Broad Institute researches provide a detailed protocol for simultaneous analysis of host and bacterial transcripts by RNA-seq. Importantly, this protocol details the steps required for efficient host and bacteria lysis, barcoding of samples, technical advances in sample preparation for low-yield sample inputs and a computational pipeline for analysis of both mammalian and microbial reads from mixed host-pathogen RNA-seq data. Sample preparation takes 3 d from cultured cells to pooled libraries. Data analysis takes an additional day. Compared with previous methods, the protocol detailed here provides a sensitive, facile and generalizable approach that is suitable for large-scale studies and will enable the field to obtain in-depth analysis of host-pathogen interactions in infection models.

Overview of the simultaneous host–pathogen RNA-seq analysis protocol

Dotted and solid lines correspond to RNA and DNA, respectively. The library is sequenced with paired-end read (R1 and R2). Inline RNA adaptors are read as part of the sequence read (R1). The P7 Illumina barcode is read from the index read (I1).