An integrated Asian human SNV and indel benchmark established using multiple sequencing methods.

Sequencing technologies have been rapidly developed recently, leading to the breakthrough of sequencing-based clinical diagnosis, but accurate and complete genome variation benchmark would be required for further assessment of precision medicine applications. Despite the human cell line of NA12878 has been successfully developed to be a variation benchmark, population-specific variation benchmark is still lacking. Here, we established an Asian human variation benchmark by constructing and sequencing a stabilized cell line of a Chinese Han volunteer. By using seven different sequencing strategies, we obtained ~3.88 Tb clean data from different laboratories, hoping to reach the point of high sequencing depth and accurate variation detection. Through the combination of variations identified from different sequencing strategies and different analysis pipelines, we identified 3.35 million SNVs and 348.65 thousand indels, which were well supported by our sequencing data and passed our strict quality control, thus should be high confidence variation benchmark. Besides, we also detected 5,913 high-quality SNVs which had 969 sites were novel and  located in the high homologous regions supported by long-range information in both the co-barcoding single tube Long Fragment Read (stLFR) data and PacBio HiFi CCS data. Furthermore, by using the long reads data (stLFR and HiFi CCS), we were able to phase more than 99% heterozygous SNVs, which helps to improve the benchmark to be haplotype level. Our study provided comprehensive sequencing data as well as the integrated variation benchmark of an Asian derived cell line, which would be valuable for future sequencing-based clinical development.

The genome sequence of Streptomyces rochei 7434AN4, which carries a linear chromosome and three characteristic linear plasmids.

Streptomyces rochei 7434AN4 produces two structurally unrelated polyketide antibiotics, lankacidin and lankamycin, and carries three linear plasmids, pSLA2-L (211 kb), -M (113 kb), and -S (18 kb), whose nucleotide sequences were previously reported. The complete nucleotide sequence of the S. rochei chromosome has now been determined using the long-read PacBio RS-II sequencing together with short-read Illumina Genome Analyzer IIx sequencing and Roche 454 pyrosequencing techniques. The assembled sequence revealed an 8,364,802-bp linear chromosome with a high G + C content of 71.7% and 7,568 protein-coding ORFs. Thus, the gross genome size of S. rochei 7434AN4 was confirmed to be 8,706,406 bp including the three linear plasmids. Consistent with our previous study, a tap-tpg gene pair, which is essential for the maintenance of a linear topology of Streptomyces genomes, was not found on the chromosome. Remarkably, the S. rochei chromosome contains seven ribosomal RNA (rrn) operons (16S-23S-5S), although Streptomyces species generally contain six rrn operons. Based on 2ndFind and antiSMASH platforms, the S. rochei chromosome harbors at least 35 secondary metabolite biosynthetic gene clusters, including those for the 28-membered polyene macrolide pentamycin and the azoxyalkene compound KA57-A.

Quality Control of Next-generation Sequencing-based In vitro Diagnostic Test for Onco-relevant Mutations Using Multiplex Reference Materials in Plasma.

Background: Widespread clinical implementation of next-generation sequencing (NGS)-based cancer in vitro diagnostic tests (IVDs) highlighted the urgency to establish reference materials which could provide full control of the process from nucleic acid extraction to test report generation. The formalin-fixed, paraffin-embedded (FFPE) tissue and blood plasma containing circulating tumor deoxyribonucleic acid (ctDNA) were mostly used for clinically detecting onco-relevant mutations. Methods: We respectively developed multiplex FFPE and plasma reference materials covering three clinically onco-relevant mutations within the epidermal growth factor receptor (EGFR) gene at serial allelic frequencies. All reference materials were quantified and validated via droplet digital polymerase chain reaction (ddPCR), and then were distributed to eight domestic manufacturers for the collaborative evaluation of the performance of several domestic NGS-based cancer IVDs covering four major NGS platforms (NextSeq, HiSeq, Ion Proton and BGISEQ). Results: All expected mutations except one at extremely low allelic frequencies were detected, despite some differences in coefficient of variation (CV) which increased with the decrease of allelic frequency (CVs ranging from 18% to 106%). It was worth noting that the CV value seemed to correlate with a particular mutation as well. The repeatability of determination of different mutations was L858R>T790M>19del. Conclusions: The results indicated our reference materials would be pivotal for quality control of NGS-based cancer IVDs and would guide the further development of reference materials covering more onco-relevant mutations.

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.

Sequencing of allotetraploid cotton (Gossypium hirsutum L. acc. TM-1) provides a resource for fiber improvement.

Upland cotton is a model for polyploid crop domestication and transgenic improvement. Here we sequenced the allotetraploid Gossypium hirsutum L. acc. TM-1 genome by integrating whole-genome shotgun reads, bacterial artificial chromosome (BAC)-end sequences and genotype-by-sequencing genetic maps. We assembled and annotated 32,032 A-subgenome genes and 34,402 D-subgenome genes. Structural rearrangements, gene loss, disrupted genes and sequence divergence were more common in the A subgenome than in the D subgenome, suggesting asymmetric evolution. However, no genome-wide expression dominance was found between the subgenomes. Genomic signatures of selection and domestication are associated with positively selected genes (PSGs) for fiber improvement in the A subgenome and for stress tolerance in the D subgenome. This draft genome sequence provides a resource for engineering superior cotton lines.

Blockage of the early step of lankacidin biosynthesis caused a large production of pentamycin, citreodiol and epi-citreodiol in Streptomyces rochei.

In our effort to find the key intermediates of lankacidin biosynthesis in Streptomyces rochei, three UV-active compounds were isolated from mutant FS18, a gene disruptant of lkcA encoding a non-ribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) hybrid enzyme. Their structures were elucidated on the basis of spectroscopic data of NMR and MS. Two compounds of a higher mobile spot on silica gel TLC (Rf=0.45 in CHCl3-MeOH=20:1) were determined to be an epimeric mixture of citreodiol and epi-citreodiol at the C-6 position in the ratio of 2:1. In contrast, the compound of a lower mobile spot (Rf=~0 in CHCl3-MeOH=20:1) was identical to a 28-membered polyene macrolide pentamycin. The yields of citreodiols and pentamycin in FS18 were 5- and 250-fold higher compared with the parent strain. Introduction of a second mutation of srrX, coding a biosynthetic gene of the signaling molecules SRBs, into mutant FS18 did not affect the production of three metabolites. Thus, their production was not regulated by the SRB signaling molecules in contrast to lankacidin or lankamycin.

The tap-tpg gene pair on the linear plasmid functions to maintain a linear topology of the chromosome in Streptomyces rochei.

Streptomyces rochei 7434AN4 carries three linear plasmids, pSLA2-L (211 kb), pSLA2-M (113 kb) and pSLA2-S (18 kb), their complete nucleotide sequences having been determined. Restriction and sequencing analysis revealed that the telomere sequences at both ends of the linear chromosome are identical to each other, are 98.5% identical to the right end sequences of pSLA2-L and pSLA2-M up to 3.1 kb from the ends and have homology to those of typical Streptomyces species. Mutant 2-39, which lost all the three linear plasmids, was found to carry a circularized chromosome. Sequence comparison of the fusion junction and both deletion ends revealed that chromosomal circularization occurred by terminal deletions followed by nonhomologous recombination. Curing of pSLA2-L from strain 51252, which carries only pSLA2-L, also resulted in terminal deletions in newly obtained mutants. The tap-tpg gene pair, which encodes a telomere-associated protein and a terminal protein for end patching, is located on pSLA2-L and pSLA2-M but has not hitherto been found on the chromosome. These results led us to the idea that the tap-tpg of pSLA2-L or pSLA2-M functions to maintain a linear chromosome in strain 7434AN4. This hypothesis was finally confirmed by complementation and curing experiments of the tap-tpg of pSLA2-M.

Whole-genome sequencing of the snub-nosed monkey provides insights into folivory and evolutionary history.

Colobines are a unique group of Old World monkeys that principally eat leaves and seeds rather than fruits and insects. We report the sequencing at 146× coverage, de novo assembly and analyses of the genome of a male golden snub-nosed monkey (Rhinopithecus roxellana) and resequencing at 30× coverage of three related species (Rhinopithecus bieti, Rhinopithecus brelichi and Rhinopithecus strykeri). Comparative analyses showed that Asian colobines have an enhanced ability to derive energy from fatty acids and to degrade xenobiotics. We found evidence for functional evolution in the colobine RNASE1 gene, encoding a key secretory RNase that digests the high concentrations of bacterial RNA derived from symbiotic microflora. Demographic reconstructions indicated that the profile of ancient effective population sizes for R. roxellana more closely resembles that of giant panda rather than its congeners. These findings offer new insights into the dietary adaptations and evolutionary history of colobine primates.

Isolation of borrelidin as a phytotoxic compound from a potato pathogenic streptomyces strain.

Streptomyces species strain GK18, isolated in Iran, induced deep-pitted lesions on potato tubers, lesions different from the raised lesions induced by the usual scab-causing phytotoxin, thaxtomin. In addition, neither thaxtomin production nor hybridization to its biosynthetic probe was detected for strain GK18, suggesting the production of a different phytotoxin. The active component was extracted with ethyl acetate from culture filtrate of strain GK18, purified by gel filtration and silica gel chromatography, and identified as an 18-membered macrolide, borrelidin, by spectroscopic analysis. The purified borrelidin induced necrosis on potato tuber slices and inhibited the growth of shoots and roots of radish seedlings. This is the first report on the phytotoxicity of borrelidin as a possible causative compound of potato scab disease.