Background. Next generation sequencing (NGS) is now being used for detecting chromosomal abnormalities in blastocyst trophectoderm (TE) cells from in v

Background: Cell‐free messenger RNA (cf‐mRNA) and long non‐coding RNA (cf‐lncRNA) are becoming increasingly important in liquid biopsy by providing biomarkers for disease prediction, diagnosis and prognosis, but the simultaneous characterization of coding and non‐coding RNAs in human biofluids remains challenging.Methods: Here, we developed polyadenylation ligation‐mediated sequencing (PALM‐Seq), an RNA sequencing strategy employing treatment of RNA with T4 polynucleotide kinase to generate cell‐free RNA (cfRNA) fragments with 5′ phosphate and 3′ hydroxyl and RNase H to deplete abundant RNAs, achieving simultaneous quantification and characterization of cfRNAs.Results: Using PALM‐Seq, we successfully identified well‐known differentially abundant mRNA, lncRNA and microRNA in the blood plasma of pregnant women. We further characterized cfRNAs in blood plasma, saliva, urine, seminal plasma and amniotic fluid and found that the detected numbers of different RNA biotypes varied with body fluids. The profiles of cf‐mRNA reflected the function of originated tissues, and immune cells significantly contributed RNA to blood plasma and saliva. Short fragments (<50 nt) of mRNA and lncRNA were major in biofluids, whereas seminal plasma and amniotic fluid tended to retain long RNA. Body fluids showed distinct preferences of pyrimidine at the 3′ end and adenine at the 5′ end of cf‐mRNA and cf‐lncRNA, which were correlated with the proportions of short fragments.Conclusion: Together, PALM‐Seq enables a simultaneous characterization of cf‐mRNA and cf‐lncRNA, contributing to elucidating the biology and promoting the application of cfRNAs.

Short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) have been already used to perform noninvasive prenatal paternity testing from maternal plasma DNA. The frequently used technologies were PCR followed by capillary electrophoresis and SNP typing array, respectively. Here, we developed a noninvasive prenatal paternity testing (NIPAT) based on SNP typing with maternal plasma DNA sequencing. We evaluated the influence factors (minor allele frequency (MAF), the number of total SNP, fetal fraction and effective sequencing depth) and designed three different selective SNP panels in order to verify the performance in clinical cases. Combining targeted deep sequencing of selective SNP and informative bioinformatics pipeline, we calculated the combined paternity index (CPI) of 17 cases to determine paternity. Sequencing-based NIPAT results fully agreed with invasive prenatal paternity test using STR multiplex system. Our study here proved that the maternal plasma DNA sequencing-based technology is feasible and accurate in determining paternity, which may provide an alternative in forensic application in the future.

Research based on a strategy of single-cell low-coverage whole genome sequencing (SLWGS) has enabled better reproducibility and accuracy for detection of copy number variations (CNVs). The whole genome amplification (WGA) method and sequencing platform are critical factors for successful SLWGS (<0.1 × coverage). In this study, we compared single cell and multiple cells sequencing data produced by the HiSeq2000 and Ion Proton platforms using two WGA kits and then comprehensively evaluated the GC-bias, reproducibility, uniformity and CNV detection among different experimental combinations. Our analysis demonstrated that the PicoPLEX WGA Kit resulted in higher reproducibility, lower sequencing error frequency but more GC-bias than the GenomePlex Single Cell WGA Kit (WGA4 kit) independent of the cell number on the HiSeq2000 platform. While on the Ion Proton platform, the WGA4 kit (both single cell and multiple cells) had higher uniformity and less GC-bias but lower reproducibility than those of the PicoPLEX WGA Kit. Moreover, on these two sequencing platforms, depending on cell number, the performance of the two WGA kits was different for both sensitivity and specificity on CNV detection. The results can help researchers who plan to use SLWGS on single or multiple cells to select appropriate experimental conditions for their applications.

Background: DNBSEQ™ platforms are new massively parallel sequencing (MPS) platforms that use DNA nanoball technology. Use of data generated from DNBSEQ™ platforms to detect single nucleotide variants (SNVs) and small insertions and deletions (indels) has proven to be quite effective, while the feasibility of copy number variants (CNVs) detection is unclear. Results: Here, we first benchmarked different CNV detection tools based on Illumina whole-genome sequencing (WGS) data of NA12878 and then assessed these tools in CNV detection based on DNBSEQ™ sequencing data from the same sample. When the same tool was used, the CNVs detected based on DNBSEQ™ and Illumina data were similar in quantity, length and distribution, while great differences existed within results from different tools and even based on data from a single platform. We further estimated the CNV detection power based on available CNV benchmarks of NA12878 and found similar precision and sensitivity between the DNBSEQ™ and Illumina platforms. We also found higher precision of CNVs shorter than 1 kbp based on DNBSEQ™ platforms than those based on Illumina platforms by using Pindel, DELLY and LUMPY. We carefully compared these two available benchmarks and found a large proportion of specific CNVs between them. Thus, we constructed a more complete CNV benchmark of NA12878 containing 3512 CNV regions. Conclusions: We assessed and benchmarked CNV detections based on WGS with DNBSEQ™ platforms and provide guidelines for future studies.