Identifying the optimal input amount and sequencing depth for B and T cell receptor repertoire profiling is challenging owing to variation in material quality and lymphocyte diversity in blood and FFPE preserved tumor specimens. Rarefaction analysis has emerged as a potential approach for assessing whether immune repertoire libraries have been sequenced to saturation. Here we present a novel automated method for saturation analysis of IGH and TCRB chain libraries derived from sequencing of peripheral blood leukocytes (PBL) and FFPE-preserved tumor RNA and DNA.
Human TCRB and IGH repertoire libraries were generated using the Oncomine TCRB and IGH assays from: (1) 25ng PBL total RNA (2) 500ng PBL gDNA (3) 150ng RNA from FFPE preserved NSCLC and (4) 200ng gDNA from FFPE preserved brain tissue. Libraries were sequenced on the Ion Torrent Gene Studio S5 then analyzed with Ion Reporter to identify clonotypes, quantify clonal expansion and diversity, and for IGH chain libraries, identify B cell clonal lineages and assess isotype usage. We then repeated clonotyping and analysis of secondary repertoire features using data that had been downsampled to fixed read depths.
We observed an asymptotic relationship between the sequencing depth and the number of B and T cell clones detected, clone Shannon diversity, and B cell clonal lineage richness and diversity, indicating that libraries had been sequenced to saturation. By contrast, T and B cell normalized Shannon entropy appeared robust to sequencing depth.
Automated downsampling analysis may serve as a convenient tool for optimizing sequencing depth and input amount for B and T cell repertoire sequencing studies. We expect this approach to become a routine component of immune repertoire analysis.
Thermo Fisher Scientific.
Thermo Fisher Scientific.
L. Quagliata: Full / Part-time employment: Thermo Fisher Scientific. T. Looney: Full / Part-time employment: Thermo Fisher Scientific. D. Topacio-Hall: Full / Part-time employment: Thermo Fisher Scientific. G. Lowman: Full / Part-time employment: Thermo Fisher Scientific.