The Role of Quality Control in Targeted Next-generation Sequencing Library Preparation

Next-generation sequencing （NGS） is getting routinely used in the diagnosis of hereditary diseases, such as human cardiomyopathies. Hence. it is of utter importance to secure high quality sequencing data, enabling the identification of disease-relevant mutations or the conclusion of negative test results. During the process of sample preparation, each protocol for target enrichment library preparation has its own requirements for quality control （QC）; however, there is little evi- dence on the actual impact of these guidelines on resulting data quality. In this study, we analyzed the impact of QC during the diverse library preparation steps of Agilent SureSelect XT target enrichment and lllumina sequencing. We quantified the parameters for a cohort of around 600 samples, which include starting amount of DNA, amount of sheared DNA, smallest and largest fragment size of the starting DNA; amount of DNA after the pre-PCR, and smallest and largest fragment size of the resulting DNA; as well as the amount of the final library, the corresponding smallest and largest fragment size, and the number of detected variants. Intriguingly, there is a high tolerance for variations in all QC steps, meaning that within the boundaries proposed in the current study, a considerable variance at each step of QC can be well tolerated without compromising NGS quality.

An integrative protocol for one‑step PCR amplicon library construction and accurate demultiplexing of pooled sequencing data

High-throughput sequencing of amplicons has been widely used to precisely and efficiently identify species compositions and analyze community structures,greatly promoting biological studies involving large amounts of complex samples,especially those involving environmental and pathogen-monitoring ones.Commercial library preparation kits for amplicon sequencing,which generally require multiple steps,including adapter ligation and indexing,are expensive and time-consuming,especially for applications at a large scale.To overcome these limitations,a“one-step PCR approach”has been previously proposed for constructions of amplicon libraries using long fusion primers.However,efficient amplifications of target genes and accurate demultiplexing of pooled sequencing data remain to be addressed.To tackle these,we present an integrative protocol for one-step PCR amplicon library construction(OSPALC).High-quality reads have been generated by this approach to reliably identify species compositions of mock bacterial communities and environmental samples.With this protocol,the amplicon library is constructed through one regular PCR with long primers,and the total cost per DNA/cDNA sample decreases to just 7%of the typical cost via the multi-step PCR approach.Empirically tested primers and optimized PCR conditions to construct OSPALC libraries for 16S rDNA V4 regions are demonstrated as a case study.Tools to design primers targeting at any genomic regions are also presented.In principle,OSPALC can be readily applied to construct amplicon libraries of any target genes using DNA or RNA samples,and will facilitate research in numerous fields.

The methodological challenge in high-throughput profiling and quantifying microRNAs

Background:MicroRNAs(miRNAs)play an essential role in various biological processes and signaling pathways through the regulation of gene expression and genome stability.Recent data indicated that the next-generation sequencing(NGS)-based high-throughput quantification of miRNAs from biofluids provided exciting possibilities for discovering biomarkers of various diseases and might help promote the development of the early diagnosis of cancer.However,the complex process of library construction for sequencing always introduces bias,which may twist the actual expression levels of miRNAs and reach misleading conclusions.Results:We discussed the deviation issue in each step during constructing miRNA sequencing libraries and suggested many strategies to generate high-quality data by avoiding or minimizing bias.For example,improvement of adapter design(a blocking element away from the ligation end,a randomized fragment adjacent to the ligation junction and UMI)and optimization of ligation conditions(a high concentration of PEG 8000,reasonable incubation temperature and time,and the selection of ligase)in adapter ligation,high-quality input RNA samples,removal of adapter dimer(solid phase reverse immobilization(SPRI)magnetic bead,locked nucleic acid(LNA)oligonucleotide,and Phi29 DNA polymerase),PCR(linear amplification,touch-down PCR),and product purification are essential factors for achieving high-quality sequencing data.Moreover,we described several protocols that exhibit significant advantages using combinatorial optimization and commercially available low-input miRNA library preparation kits.Conclusions:Overall,our work provides the basis for unbiased high-throughput quantification of miRNAs.These data will help achieve optimal design involving miRNA profiling and provide reliable guidance for clinical diagnosis and treatment by significantly increasing the credibility of potential biomarkers.