PALM-Seq: integrated sequencing of cell-free long RNA and small RNA

Abstract

Cell-free RNA, including both long RNA and small RNA, has been considered important for its biological functions and potential clinical usage, but the major challenge is to effectively sequence them at the same time. Here we present PolyAdenylation Ligation Mediated-Seq (PALM-Seq), an integrated sequencing method for cell-free long and small RNA. Through terminal modification and addition of 3' polyadenylation and 5' adaptor, we could get mRNA, long non-coding RNA, microRNA, tRNA, piRNA and other RNAs in a single library. With target RNA depletion, all these RNAs could be sequenced with relatively low depth. Using PALM-Seq, we identified pregnant-related mRNAs, long non-coding RNAs and microRNAs in female plasma. We also applied PALM-Seq to sequence RNA from amniotic fluids, leukocytes and placentas, and could find RNA signatures associated with specific sample type. PALM-Seq provides an integrated, cost-effective and simple method to characterize the landscape of cell-free RNA, and can stimulate further progress in cell-free RNA study and usage.

Plasma cell-free RNA characteristics in COVID-19 patients

“…Each platform has its unique advantages and disadvantages. Here, we adopt PALM-seq, which has been compared with multiple public data produced by SMARTer Seq, ScriptSeq, and small RNA-seq in our previous report (Yang et al 2019). Compared with the traditional RNA library construction that requires a lot of blood, it is difficult to prepare large-scale cfRNA libraries with the high cost for large-scale cfRNA library preparation and low mapping rate.…”

“…Serial plasma was collected from eight healthy donors and 37 COVID-19 patients (19 mild and 18 severe patients) and used for cfRNAs sequencing by PALM-seq (Fig. 1A; Supplemental Table S1; Yang et al 2019). Based on the clinical symptoms, two distinct subgroups of COVID-19 patients were enrolled, including the mild and severe groups.…”

Impact of blood storage conditions on the transcript profile of plasma cell-free RNA

“…The transcriptome library was constructed using PolyAdenylation Ligation Mediated-Seq (PALM-Seq) method [9], which is a simultaneous sequencing method for cell-free coding and non-coding RNA. cfRNA sequencing was performed on BGISEQ-500RS (single-end 100 bp) platform with an average depth of 100 million reads per sample.…”

“…Details of cfRNA sequencing data preprocess were carried out as previously described [9]. In brief, adapter and low-quality reads were trimmed, then reads with > 10% ‘N’ base or shorter than 17bp were filtered by cutadapt [12].…”

“…Yet no study has systematically evaluated the influence of blood storage conditions on the full transcriptome of cfRNA in plasma. With the development of sequencing technology of plasma cfRNA [9, 10], more informative cfRNA can be detected, which makes it possible to obtain the whole transcriptome profile of plasma cfRNA to evaluate the impact of the delay of blood processing.…”