Rapid identification of full-length genome and tracing variations of monkeypox virus in clinical specimens based on mNGS and amplicon sequencing.

The monkeypox virus (MPXV) has triggered a current outbreak globally. Genome sequencing of MPXV and rapid tracing of genetic variants will benefit disease diagnosis and control. It is a significant challenge but necessary to optimize the strategy and application of rapid full-length genome identification and to track variations of MPXV in clinical specimens with low viral loads, as it is one of the DNA viruses with the largest genome and the most AT-biased, and has a significant number of tandem repeats. Here we evaluated the performance of metagenomic and amplicon sequencing techniques, and three sequencing platforms in MPXV genome sequencing based on multiple clinical specimens of five mpox cases in Chinese mainland. We rapidly identified the full-length genome of MPXV with the assembly of accurate tandem repeats in multiple clinical specimens. Amplicon sequencing enables cost-effective and rapid sequencing of clinical specimens to obtain high-quality MPXV genomes. Third-generation sequencing facilitates the assembly of the terminal tandem repeat regions in the monkeypox virus genome and corrects a common misassembly in published sequences. Besides, several intra-host single nucleotide variations were identified in the first imported mpox case. This study offers an evaluation of various strategies aimed at identifying the complete genome of MPXV in clinical specimens. The findings of this study will significantly enhance the surveillance of MPXV.

Performance comparison of NextSeq and Ion Proton platforms for molecular diagnosis of clinical oncology.

PURPOSE: Next-generation sequencing is a powerful approach to detect genetic mutations with which cancer diagnosis and treatment can be tailored to the individual patient in the era of personalized and precision medicine. Ion Torrent Systems Ion Proton and Illumina NextSeq are 2 major targeted sequencing platforms; however, not much work has been done to compare these platforms' performance for mutation detection in formalin-fixed paraffin-embedded (FFPE) materials. METHODS: We benchmarked the performance by using a collection of FFPE samples from 23 patients with different cancers for NextSeq and Ion Proton platforms. We report analysis of sequencing in terms of average coverage depth, read length, and variant detection. RESULTS: Sequencing results by NextSeq and Ion Proton displayed near perfect coverage behavior (>99%) on target region. We analyzed the ability to call variants from each platform and found that Ion Proton sequencing can identify 89% of single nucleotide variants (SNVs) whose mutant allele frequency (MAF) is greater than or equal to 5% detected by the NextSeq pipeline in common analytical regions. The correlation coefficient of MAF for those common SNVs was 1.0046 (R2 = 0.973) between the 2 platforms. To call lower mutant frequency (5%-10%) mutations for NextSeq sequencing, coverage depth should be improved. The concordance of small insertions and deletions between these 2 pipelines was up to 100%. CONCLUSIONS: The 2 sequencing pipelines evaluated were able to generate usable sequence and had high concordance. They are proper for mutation detection in clinical application.

Detection of circulating tumor DNA in patients with advanced non-small cell lung cancer.

Circulating tumor DNA (ctDNA) isolated from plasma has great potential in identification of gene mutation in non-small cell lung cancers (NSCLC), which is a non-invasive technique and can avoid the inherent shortcomings of tissue biopsy. However the ability of NGS to detect gene mutation in plasma ctDNA has not been broadly explored. To assess the diagnostic ability of ctDNA for the total mutation profile, including single nucleotide variations (SNVs), insertions and deletions (indels) and gene rearrangements, we performed a targeted DNA sequencing approach to screen NSCLC related driver gene mutations in both tissue biopsies and matched blood plasma samples from 39 advanced NSCLC patients from China. The sensitivity of EGFR, KRAS, PIK3CA mutations and gene rearrangements detected in plasma ctDNA was 70.6%, 75%, 50% and 60%, respectively and the overall concordance of gene mutations between tissue DNA and plasma ctDNA was 78.21%. Our data provide evidence that ctDNA in plasma is likely to become an alternative source for cancer-related mutations profiling in advanced NSCLC patients and targeted sequencing of ctDNA offers a promising perspective on precise diagnostics and may serve as a feasible option for clinical monitoring of NSCLC patients.

Development and validation of an ultra-high sensitive next-generation sequencing assay for molecular diagnosis of clinical oncology.

Dramatic improvements in the understanding of oncogenes have spurred the development of molecular target therapies, which created an exigent need for comprehensive and rapid clinical genotyping. Next-generation sequencing (NGS) assay with increased performance and decreased cost is becoming more widely used in clinical diagnosis. However, the optimization and validation of NGS assay remain a challenge, especially for the detection of somatic variants at low mutant allele fraction (MAF). In the present study, we developed and validated the Novogene Comprehensive Panel (NCP) based on targeted capture for NGS analysis. Due to the high correlation between SNV/INDEL detection performance and target coverage, here we focused on these two types of variants for our deep sequencing strategy. To validate the capability of NCP in single-nucleotide variant (SNV) and small insert and deletion (INDEL) detection, we implemented a practical validation strategy with pooled cell lines, deep sequencing of pooled samples (>2000X average unique coverage across target region) achieving >99% sensitivity and high specificity (positive predictive value, PPV >99%) for all types of variations with expected MAF >5%. Furthermore, given the high sensitivity and that false positive may exist in this assay, we confirmed its accuracy of variants with MAF <5% using 35 formalin-fixed and paraffin-embedded (FFPE) tumor specimens by Quantstudio 3D Digital PCR (dPCR; Life Technologies) and obtained a high consistency (32 of 35 mutations detected by NGS were verified). We also used the amplification refractory mutation system (ARMS) to verify the variants with a MAF in a broad range of 2-63% detected in 33 FFPE samples and reached a 100% PPV for this assay. As a potential clinical diagnosis tool, NCP can robustly and comprehensively analyze clinical-related genes with high sensitivity and low cost.

Population Genomics Reveals Low Genetic Diversity and Adaptation to Hypoxia in Snub-Nosed Monkeys.

Snub-nosed monkeys (genus Rhinopithecus) are a group of endangered colobines endemic to South Asia. Here, we re-sequenced the whole genomes of 38 snub-nosed monkeys representing four species within this genus. By conducting population genomic analyses, we observed a similar load of deleterious variation in snub-nosed monkeys living in both smaller and larger populations and found that genomic diversity was lower than that reported in other primates. Reconstruction of Rhinopithecus evolutionary history suggested that episodes of climatic variation over the past 2 million years, associated with glacial advances and retreats and population isolation, have shaped snub-nosed monkey demography and evolution. We further identified several hypoxia-related genes under selection in R. bieti (black snub-nosed monkey), a species that exploits habitats higher than any other nonhuman primate. These results provide the first detailed and comprehensive genomic insights into genetic diversity, demography, genetic burden, and adaptation in this radiation of endangered primates.

Molecular traces of alternative social organization in a termite genome.

Although eusociality evolved independently within several orders of insects, research into the molecular underpinnings of the transition towards social complexity has been confined primarily to Hymenoptera (for example, ants and bees). Here we sequence the genome and stage-specific transcriptomes of the dampwood termite Zootermopsis nevadensis (Blattodea) and compare them with similar data for eusocial Hymenoptera, to better identify commonalities and differences in achieving this significant transition. We show an expansion of genes related to male fertility, with upregulated gene expression in male reproductive individuals reflecting the profound differences in mating biology relative to the Hymenoptera. For several chemoreceptor families, we show divergent numbers of genes, which may correspond to the more claustral lifestyle of these termites. We also show similarities in the number and expression of genes related to caste determination mechanisms. Finally, patterns of DNA methylation and alternative splicing support a hypothesized epigenetic regulation of caste differentiation.

Whole-genome sequencing of Berkshire (European native pig) provides insights into its origin and domestication.

Domesticated organisms have experienced strong selective pressures directed at genes or genomic regions controlling traits of biological, agricultural or medical importance. The genome of native and domesticated pigs provide a unique opportunity for tracing the history of domestication and identifying signatures of artificial selection. Here we used whole-genome sequencing to explore the genetic relationships among the European native pig Berkshire and breeds that are distributed worldwide, and to identify genomic footprints left by selection during the domestication of Berkshire. Numerous nonsynonymous SNPs-containing genes fall into olfactory-related categories, which are part of a rapidly evolving superfamily in the mammalian genome. Phylogenetic analyses revealed a deep phylogenetic split between European and Asian pigs rather than between domestic and wild pigs. Admixture analysis exhibited higher portion of Chinese genetic material for the Berkshire pigs, which is consistent with the historical record regarding its origin. Selective sweep analyses revealed strong signatures of selection affecting genomic regions that harbor genes underlying economic traits such as disease resistance, pork yield, fertility, tameness and body length. These discoveries confirmed the history of origin of Berkshire pig by genome-wide analysis and illustrate how domestication has shaped the patterns of genetic variation.

Ground tit genome reveals avian adaptation to living at high altitudes in the Tibetan plateau.

The ground tit (Parus humilis) is endemic to the Tibetan plateau. It is a member of family Paridae but it was long thought to be related to the ground jays because of their morphological similarities. Here we present the ground tit's genome and re-sequence two tits and one ground jay, to clarify this controversially taxonomic status and uncover its genetic adaptations to the Tibetan plateau. Our results show that ground tit groups with two tits and it diverges from them between 7.7 and 9.9 Mya. Compared with other avian genomes, ground tit shows expansion in genes linked to energy metabolism and contractions in genes involved in immune and olfactory perception. We also found positively selected and rapidly evolving genes in hypoxia response and skeletal development. These results indicated that ground tit evolves basic strategies and 'tit-to-jay' change for coping with the life in an extreme environment.