Identification of individuals by RNA sequencing of low template samples

In this pilot study, we made dilutions of DNA/RNA co-extractions (1000–12.5 pg gDNA) and whole transcriptome shotgun sequenced RNA on a NovaSeq 6000 platform (Illumina). Whole blood samples from two individuals were sequenced in triplicate for each dilution. More than 450,000 RNA variants were identified to have overlapping positions with the gnomAD 3.0 DNA variant database in the 50 genes with most reads. Between 7000 and 45,000 of these loci were covered with at least 70 reads in the various dilutions. A total of 171 SNPs with global minor allele frequencies > 0.1 were identified in all dilutions with RNA input equivalent to 12.5 pg gDNA. Of these, 56 were genotyped with the Infinium Omni5–4 SNP-chip (Illumina) using DNA from the two individuals and 53 SNP genotypes in 22 autosomal genes were confirmed between RNA and DNA. The mean match probability based on the most polymorphic SNP in the 22 genes was 5 × 10−8. Thus, shotgun RNA sequencing of low template samples may provide both human identification and tissue identification.