OR45 RNA sequencing of two lymphoblastoid cell lines reveals novel microrna transcripts of the MHC

Abstract

Aim MicroRNA (miRNA) are short (∼22 bp) single stranded RNA transcripts that bind to and regulate the posttranscriptional expression of targeted mRNA transcripts. There are 12 known miRNA transcribed from within the MHC, which have been previously identified by RNA sequencing of various primary cell types. This approach to identify novel miRNA is however inherently flawed since it relies upon significant sequence concordance between the reference genome and the generated reads, which is particularly problematic for transcripts originating from within the highly polymorphic MHC. To address this gap, we sequenced short RNA transcripts (less than 50 bp) from two homozygous cell lines with fully characterized MHC haplotypes, PGF and COX, in order to identify and characterize novel microRNA (miRNA) transcripts from within the MHC. Methods Total RNA was extracted from two lymphoblastoid cell lines, PGF and COX. Size selected transcripts (less thank 50 bp) were isolated for library construction and sequenced on an Illumina HiSeq 2500. Single-end reads from PGF and COX were mapped to their respective MHC haplotype reference sequence using NovoAlign. Novel miRNA were identified using a custom computational analysis pipeline and validated by qPCR. Results In total, our analysis has identified 67 and 47 high confidence, significantly expressed, novel miRNAs from RNA-Seq data generated from PGF and COX lymphoblastoid cell lines, respectively. Of these, 25 identified novel miRNA were found to be expressed in both PGF and COX cells, while 42 and 22 novel miRNA were found to exhibit haplotype specific expression patterns in PGF and COX, respectively. Conclusions The ability to de novo assemble the MHC is critical to ensure accurate RNA-Seq read mapping within the highly variable MHC. Our results demonstrate the utility of an accurate MHC reference assembly in combination with a custom developed computational analysis pipeline to identify and characterize novel miRNA transcripts from within the MHC of two homozygous cell lines with fully characterized MHC reference sequences. Our analysis revealed haplotype specific as well as commonly expressed novel miRNA transcripts, identifying novel regulatory elements encoded within the MHC.