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.

Comprehensive validation of a diagnostic strategy for sequencing genes with one or multiple pseudogenes using pseudoxanthoma elasticum as a model

Pseudogenes are frequently encountered noncoding sequences with a high sequence similarity to their protein-coding paralogue.For this reason,their presence is often considered troublesome in molecular diagnostics.In pseudoxanthoma elasticum(PXE),a disease predominantly caused by mutations in ATPbinding cassette family C member 6(ABCC6),the presence of two pseudogenes complicates the analysis of sequence data.With whole-exome sequencing(WES)becoming the standard of care in molecular diagnostics,we wanted to evaluate whether this technique is as reliable as gene-specific targeted enrichment analysis for the analysis of ABCC6.We established a PCR-based targeted enrichment and next-generation sequencing testing approach and demonstrated that the ABCC6-specific enrichment combined with the applied mapping algorithm overcomes the complication of ABCC6 pseudogene aspecificities,contrary to WES.We propose a time-and cost-efficient diagnostic strategy for comprehensive and accurate molecular genetic testing of PXE,which is highly automatable.

The impact of next-generation sequencing on genomics

This article reviews basic concepts, general applications, and the potential impact of next-generation sequencing （NGS） technologies on genomics, with particular reference to currently available and possible future platforms and bioinformatics. NGS technologies have demon- strated the capacity to sequence DNA at unprecedented speed, thereby enabling previously unimaginable scientific achievements and novel biological applications. But, the massive data produced by NGS also presents a significant challenge for data storage, analyses, and management solutions. Advanced bioinformatic tools are essential for the successful application of NGS technology. As evidenced throughout this review, NGS technologies will have a striking impact on genomic research and the entire biological field. With its ability to tackle the unsolved challenges unconquered by previous genomic technologies, NGS is likely to unravel the complexity of the human genome in terms of genetic variations, some of which may be confined to susceptible loci for some common human conditions. The impact of NGS technologies on genomics will be far reaching and likely change the field for years to come.

Porphyrin-based Nanosonosensitizers Combined with Targeting Peptides for Sonodynamic Therapy of Glioma

Traditional cancer treatments have disadvantages of large trauma area and toxic side effects while killing cancer cells.Peptide-targeted sonodynamic therapy(SDT)can effectively improve specificity of cancer treatment and overcome the problem of low tissue penetration depth caused by a photo-driven therapy.Herein,we developed a porphyrin-based sonosensitizer with a water-soluble polymer as a biological carrier and a cRGD peptide for tumor targeting,which constituted a nano sonosensitizer(T-cRGD NPs)for fluorescence imaging-guided sonodynamic therapy.A comparable sonosensitizer(T-PEG NPs)without the targeting unit was also prepared for illustration of therapeutic performance.Attribute to the role of peptide targeting,T-cRGD NPs can accumulate and enter tumor cells for fluorescence imaging and show a superior SDT effect than T-PEG NPs in vitro.The imaging in vivo reveals that T-cRGD NPs can enrich in tumor tissues within 14 h with a good biocompatibility.