Development and characterization of novel polymorphic microsatellite markers for the Korean freshwater snail Semisulcospira coreana and cross-species amplification using next-generation sequencing

Korean freshwater snails of the genus Semisulcospira are widely distributed across East Asia.It has been a very popular nutritional food in Korea,and is an ecologically important water quality indicator because it lives only in clean water.However,no microsatellite markers have been generated to study the population genetic diversity of this genus.In the present study,we developed and characterized 18 novel microsatellite loci from Semisulcospira coreana genomic DNA.The microsatellites were isolated using 454 GS-FLX titanium sequencing and 18 markers were used for genotyping in S.coreana.In addition,we also tested the cross-species transferability of the microsatellite markers in four additional Semisulcospira spp.We identified 18 polymorphic loci and the number of alleles per loci,and their polymorphism information content values ranged from 2 to 17 and 0.203 to 0.902,respectively.The observed and expected heterozygosities of the loci ranged from 0.063 to 0.924 and 0.226 to 0.924,respectively.According to the analysis of the cross-species transferability of these markers,four species,S.forticosta,S.gottschei,S.tegulata,and S.libertina,showed a very high transferability(80%–85%).These results show that this set of nuclear markers could be useful for population genetics studies of this species and closely related species.

Phytoplankton diversity in a tropical bay,North Borneo,Malaysia as revealed by light microscopy and Next-Generation Sequencing

Assessments of phytoplankton diversity in Sabah waters,North Borneo,have primarily relied on morphology-based identification,which has inherent biases and can be time-consuming.Next-Generation Sequencing(NGS)technology has been shown to be capable of overcoming several limitations of morphology-based methods.Samples were collected from the Sepanggar Bay over the course of the year 2018 in different monsoon seasons.Morphology-based identification and NGS sequencing of the V8–V9 region of the 18S LSU rDNA were used to investigate the diversity of the phytoplankton community.Microscopy and NGS showed complementary results with more diatom taxa detected by microscopy whereas NGS detected smaller and rarer taxa.The harmful algal genera in the study site comprised of Skeletonema,Margalefidinium,Pyrodinium,Takayama,and Alexandrium as detected by NGS.This study showed that that an integrative approach of both morphological and molecular techniques could provide more comprehensive information about the phytoplankton community as the approach captured quantitative variability as well as the diversity of phytoplankton species.

Trastuzumab,not lapatinib,has therapeutic effects on Chinese patients with HER2-positive cholangiocarcinoma

As a relatively uncommon orphan tumor with high mortality,biliary tract cancer(BTC)presents an aggressive course and heterogeneous clinical features[1].BTC patients present with advanced manifestations[2].Unfortunately,there has been little progress in the management of BTC.Most patients have inoperable lesions and must receive palliative therapy.Gemcitabine-based chemotherapy has been the only widely accepted first-line treatment for advanced BTC[3].Nevertheless,BTCs are often refractory to chemotherapeutic regimens,leading to a poor clinical outcome in these patients.Recently,with the rapid development of next generation sequencing(NGS)technologies,some actionable mutations such as those in IDH1,FGFR2,BRAF,HER2 genes,and unique molecular subsets in BTCs have been identified[4],and related targeted therapy against actionable mutations has been introduced into clinical practice as a promising therapeutic strategy[5].

Identification, characterization and full-length sequence analysis of a novel endornavirus in common sunflower (Helianthus annuus L.)

To identify the possible quarantine viruses in seven common sunflower varieties imported from the United States of America and the Netherlands, we tested total RNAs extracted from the leaf tissues using next-generation sequencing of small RNAs. After analysis of small RNA sequencing data, no any quarantine virus was found, but a double-stranded RNA（dsRNA） molecule showing typical genomic features of endornavirus was detected in two varieties, X3939 and SH1108. Full-length sequence and phylogenetic analysis showed that it is a novel endornavirus, temporarily named as Helianthus annuus alphaendornavirus（HaEV）. Its full genome corresponds to a 14 662-bp dsRNA segment, including a 21-nt 5′ untranslated region（UTR）, 3＇ UTR ending with the unique sequence CCCCCCCC and lacking a poly（A） tail. An open reading frame（ORF） that encodes a deduced 4 867 amino acids（aa） polyprotein with three domains： RdRP, Hel and UGT（UDP-glycosyltransferase）. HaEV mainly distributed in the cytoplasm but less in the nucleus of leaf cells by fluorescence in situ hybridization（FISH） experiment. This virus has a high seed infection rate in the five varieties, X3907, X3939, A231, SH1108 and SR1320. To our knowledge, this is the first report about the virus of the family Endornaviridae in the common sunflower.

Genome re-sequencing analysis uncovers pathogenecity-related genes undergoing positive selection in Magnaporthe oryzae

Rice blast caused by Magnaporthe oryzae （M. oryzae） is one of the most destructive diseases, which causes significant rice yield losses and affects global food security. To better understand genetic variations among different isolates of M. oryzae in nature, we re-sequenced the genomes of two field isolates, CH43 and Zhong-10-8-14, which showed distinct pathogenecity on most of the rice cultivars. Genome-wide genetic variation analysis reveals that ZHONG-10-8-14 exhibits higher sequence variations than CH43. Structural variations （SVs） detection shows that the sequence variations primarily occur in exons and intergenic regions. Bioinformatics analysis for gene variations reveals that many pathogenecity-related pathways are enriched. In addition, 193 candidate effectors with various DNA polymorphisms were identified, including two known effectors AVR-Pik and AVR-Pital. Comparative polymorphism analysis of thirteen randomly selected effectors suggests that the genetic variations of effectors are under positive selection. The expression pattern analysis of several pathogenecity-related variant genes indicates that these genes are differentially regulated in two isolates, with much higher expression levels in Zhong-10-8-14 than CH43. Our data demonstrate that the genetic variations of effectors and pathogenecity-related genes are under positive selection, resulting in the distinct pathogeuicities of CH43 and Zhong- 10-8-14 on rice.

Next generation sequencing of Apis mellifera syriaca identifies genes for Varroa resistance and beneficial bee keeping traits

Apis mellifera syriaca exhibits a high degree of tolerance to pests and pathogens including varroa mites. This native honey bee subspecies of Jordan expresses behavioral adaptations to high temperature and dry seasons typical of the region. However, persistent honey bee imports of commercial breeder lines are endangering local honey bee population. This study reports the use of next-generation sequencing （NGS） technology to study the A. m. syriaca genome and to identify genetic factors possibly contributing toward mite resistance and other favorable traits. We obtained a total of 46.2 million raw reads by applying the NGS to sequence A. m. syriaca and used extensive bioinformatics approach to identify several candidate genes for Varroa mite resistance, behavioral and immune responses char- acteristic for these bees. As a part of characterizing the functional regulation of molecular genetic pathway, we have mapped the pathway genes potentially involved using information from Drosophila melanogaster and present possible functional changes implicated in responses to Varroa destructor mite infestation toward this. We performed in-depth functional annotation methods to identify -600 candidates that are relevant, genes involved in pathways such as microbial recognition and phagocytosis, peptidoglycan recognition protein family, Gram negative binding protein family, phagocytosis receptors, serpins, Toll signaling pathway, Imd pathway, Tnf, JAK-STAT and MAPK pathway, heamatopioesis and cellular response pathways, antiviral, RNAi pathway, stress factors, etc. were selected. Finally, we have cataloged function-specific polymorphisms between A. mellifera and A. m. syriaca that could give better understanding of varroa mite resistance mechanisms and assist in breeding. We have identified immune related embryonic development （Cactus, Relish, dorsal, Ank2, baz）, Varroa hygiene （NorpA2, Zasp, LanA, gasp, impl3） and Varroa resistance （Pug, pcmt, elk, elf3-s10, Dscam2, Dhc64C, gro, futsch） functional variations genes between A. mellifera and A. m. syriaca that could be used to develop an effective molecular tool for bee conservation and breeding programs to improve locally adapted strains such as syriaca and utilize their advantageous traits for the benefit of apiculture industry.

Next generation sequencing under de novo genome assembly

The next generation sequencing （NGS） is an important process which assures inexpen- sive organization of vast size of raw sequence dataset over any traditional sequencing systems or methods. Various aspects of NGS such as template preparation, sequencing imaging and genome alignment and assembly outline the genome sequencing and align- ment. Consequently, de Bruijn graph （dBG） is an important mathematical tool that graphically analyzes how the orientations are constructed in groups of nucleotides. Basi- cally, dBG describes the formation of the genome segments in circular iterative fashions. Some pivotal dBG-based de novo algorithms and software packages such as T-IDBA, Oases, IDBA-tran, Euler, Velvet, ABYSS, AllPaths, SOAPde novo and SOAPde novo2 are illustrated in this paper. Consequently, overlap layout consensus （OLC） graph-based algorithms also play vital role in NGS assembly. Some important OLC-based algorithms such as MIRA3, CABOG, Newbler, Edena, Mosaik and SHORTY are portrayed in this paper. It has been experimented that greedy graph-based algorithms and software pack- ages are also vital for proper genome dataset assembly. A few algorithms named SSAKE, SHARCGS and VCAKE help to perform proper genome sequencing.

Next-generation sequencing and single-cell RT-PCR reveal a distinct variable gene usage of porcine antibody repertoire following PEDV vaccination

Porcine epidemic diarrhea virus(PEDV)is the most common diarrhea-causing pathogen in newborn piglets.The clarifications of the overall antibody repertoire and antigen-specific antibody repertoire are essential to provide important insights into the B-cell response and reshape new vaccines.Here,we applied next-generation sequencing(NGS)technology to investigate immunoglobulin(Ig)variable(V)gene segment usage of swine B-cells from peripheral blood lymphocytes(PBL)and mesenteric lymph node(MLN)cells following PEDV vaccination.We identified the transcripts of all functional Ig V-genes in antibody repertoire.IgHV1 S2,IgKV1-11,and IgLV3-4 were the most prevalent gene segments for heavy,kappa,and lambda chains,respectively,in PBL and MLN.Unlike previous studies,IgKV1,instead of IgKV2,and IgLV3,instead of IgLV8,were the prevalent Ig V-gene families for kappa and lambda light chains,respectively.We further examined the antibody repertoire of PEDV spike-specific B cells by single-cell RT-PCR.In contrast to the overall antibody repertoire,Ig V-gene segments of PEDV spike-specific B cells preferentially adopted IgHV1-4 and IgHV1-14 for heavy chain,IgKV1-11 for kappa chain,and IgLV3-3 for lambda chain.These results represent a comprehensive analysis to characterize the Ig V-gene segment usage in the overall and PEDV spike-specific antibody repertoire in PBL and MLN.

Transcriptomics:Advances and approaches

Transcriptomics is one of the most developed fields in the post-genomic era.Transcriptome is the complete set of RNA transcripts in a specific cell type or tissue at a certain developmental stage and/or under a specific physiological condition,including messenger RNA,transfer RNA,ribosomal RNA,and other non-coding RNAs.Transcriptomics focuses on the gene expression at the RNA level and offers the genome-wide information of gene structure and gene function in order to reveal the molecular mechanisms involved in specific biological processes.With the development of next-generation high-throughput sequencing technology,transcriptome analysis has been progressively improving our understanding of RNA-based gene regulatory network.Here,we discuss the concept,history,and especially the recent advances in this inspiring field of study.

Single Nucleotide Polymorphism Identification in Polyploids： A Review, Example, and Recommendations

Understanding the relationship between genotype and phenotype is a major biological question and being able to predict phenotypes based on molecular genotypes is integral to molecular breeding. Whole- genome duplications have shaped the history of all flowering plants and present challenges to elucidating the relationship between genotype and phenotype, especially in neopolyploid species. Although single nucleotide polymorphisms （SNPs） have become popular tools for genetic mapping, discovery and appli- cation of SNPs in polyploids has been difficult. Here, we summarize common experimental approaches to SNP calling, highlighting recent polyploid successes. To examine the impact of software choice on these analyses, we called SNPs among five peanut genotypes using different alignment programs （BWA-mem and Bowtie 2） and variant callers （SAMtools, GATK, and Freebayes）. Alignments produced by Bowtie 2 and BWA-mem and analyzed in SAMtools shared 24.5% concordant SNPs, and SAMtools, GATK, and Freebayes shared 1.4% concordant SNPs. A subsequent analysis of simulated Brassica napus chromosome 1A and 1C genotypes demonstrated that, of the three software programs, SAMtools performed with the highest sensitivity and specificity on Bowtie 2 alignments. These results, however, are likely to vary among species, and we therefore propose a series of best practices for SNP calling in polyploids.

Metagenomics: Facts and Artifacts, and Computational Challenges

Metagenomics is the study of microbial communities sampled directly from their natural environment, without prior culturing. By enabling an analysis of populations including many （so-far） unculturable and often unknown microbes, metagenomics is revolutionizing the field of microbiology, and has excited researchers in many disciplines that could benefit from the study of environmental microbes, including those in ecology, environmental sciences, and biomedicine. Specific computational and statistical tools have been developed for metagenomic data analysis and comparison. New studies, however, have revealed various kinds of artifacts present in metagenomics data caused by limitations in the experimental protocols and/or inadequate data analysis procedures, which often lead to incorrect conclusions about a microbial community. Here, we review some of the artifacts, such as overestimation of species diversity and incorrect estimation of gene family frequencies, and discuss emerging computational approaches to address them. We also review potential challenges that metagenomics may encounter with the extensive application of next-generation sequencing （NGS） techniques.

Mutation profile and its correlation with clinicopathology in Chinese hepatocellular carcinoma patients

Background:Hepatocellular carcinoma(HCC)is one of the most common causes of cancer worldwide.Although many studies have focused on oncogene characteristics,the genomic landscape of Chinese HCC patients has not been fully clarified.Methods:A total of 165 HCC patients,including 146 males and 19 females,were enrolled.The median age was 55 years(range,27-78 years).Corresponding clinical and pathological information was collected for further analysis.A total of 168 tumor tissues from these patients were selected for next-generation sequencing(NGS)-based 450 panel gene sequencing.Genomic alterations including single nucleotide variations(SNV),short and long insertions and deletions(InDels),copy number variations,and gene rearrangements were analyzed.Tumor mutational burden(TMB)was measured by an algorithm developed in-house.The top quartile of HCC was classified as TMB high.Results:A total of 1,004 genomic alterations were detected from 258 genes in 168 HCC tissues.TMB values were identified in 160 HCC specimens,with a median TMB of 5.4 Muts/Mb(range,0-28.4 Muts/Mb)and a 75%TMB of 7.7 Muts/Mb.The most commonly mutated genes were TP53,TERT,CTNNB1,AXIN1,RB1,TSC2,CCND1,ARID1A,and FGF19.SNV was the most common mutation type and C:G>T:A and guanine transformation were the most common SNVs.Compared to wild-type patients,the proportion of Edmondson grade III-IV and microvascular invasion was significantly higher in TP53 mutated patients(P<0.05).The proportion of tumors invading the hepatic capsule was significantly higher in TERT mutated patients(P<0.05).The proportion of Edmondson grade I-II,alpha fetoprotein(AFP)<25μmg/L,and those without a history of hepatitis B was significantly higher in CTNNB1 mutated patients(P<0.05).CTNNB1 mutations were associated with TMB high in HCC patients(P<0.05).Based on correlation analysis,the mutation of TP53 was independently correlated with microvascular invasion(P=0.002,OR=3.096)and Edmondson grade III-IV(P=0.008,OR=2.613).The mutation of TERT was independently correlated with tumor invasion of the liver capsule(P=0.001,OR=3.030),and the mutation of CTNNB1 was independently correlated with AFP(<25μmg/L)(P=0.009,OR=3.414).Conclusions:The most frequently mutated genes of HCC patients in China were TP53,TERT,and CTNNB1,which mainly lead to the occurrence and development of HCC by regulating the P53 pathway,Wnt pathway,and telomere repair pathway.There were more patients with microvascular invasion and Edmondson III-IV grade in TP53 mutated patients and more patients with hepatic capsule invasion in TERT mutated patients,while in CTNNB1 mutated patients,there were more patients with Edmondson I-II grade,AFP<25μmg/L,and a non-hepatitis B background.Also,the TMB values were significantly higher in CTNNB1 mutated patients than in wild type patients.

Dawn of ocular gene therapy:implications for molecular diagnosis in retinal disease

Personalized medicine aims to utilize genomic information about patients to tailor treatment. Gene replacement therapy for ra- re genetic disorders is perhaps the most extreme form of personalized medicine, in that the patients＇ genome wholly determines their treatment regimen. Gene therapy for retinal disorders is poised to become a clinical reality. The eye is an optimal site for gene therapy due to the relative ease of precise vector delivery, immune system isolation, and availability for monitoring of any potential damage or side effects. Due to these advantages, clinical trials for gene therapy of retinal diseases are currently underway. A necessary precursor to such gene therapies is accurate molecular diagnosis of the mutation（s） underlying disease. In this review, we discuss the application of Next Generation Sequencing （NGS） to obtain such a diagnosis and identify disease causing genes, using retinal disorders as a case study. After reviewing ocular gene therapy, we discuss the application of NGS to the identification of novel Mendelian disease genes. We then compare current, array based mutation detection methods against next NGS-based methods in three retinal diseases： Leber＇s Congenital Amaurosis, Retinitis Pigmentosa, and Stargardt＇s disease. We conclude that next-generation sequencing based diagnosis offers several advantages over array based methods, including a higher rate of successful diagnosis and the ability to more deeply and efficiently assay a broad spectrum of mutations. However, the relative difficulty of interpreting sequence results and the development of standardized, reliable bioinforrnatic tools remain outstanding concerns. In this review, recent advances NGS based molecular diagnoses are discussed, as well as their implications for the development of personalized medicine.

Recent advances in elucidating the genetics of common variable immunodeficiency

Common variable immunodeficiency disorders(CVID),a heterogeneous group of inborn errors of immunity,is the most common symptomatic primary immunodeficiency disorder.Patients with CVID have highly variable clinical presentation.With the advent of whole genome sequencing and genome wide association studies(GWAS),there has been a remarkable improvement in understanding the genetics of CVID.This has also helped in understanding the pathogenesis of CVID and has drastically improved the management of these patients.A multiomics approach integrating the DNA sequencing along with RNA sequencing,proteomics,epigenetic and metabolomics profile is the need of the hour to unravel specific CVID associated disease pathways and novel therapeutic targets.In this review,we elaborate various techniques that have helped in understanding the genetics of CVID.

Investigation on the genetic-inconsistent paternity cases using the MiSeq FGx system

Mutations might challenge the paternity index calculation in forensic identification.While many studies have focussed on the autosomal short tandem repeats(A-STR),the mutation status of sex chromosomes and single nucleotide polymorphism(SNP)remain blank.Next generation sequencing(NGS),known as high throughput and large sequence polymorphism,is a promising tool for forensic genetics.To describe the mutation landscapes in the paternity cases with genetic inconsistencies,a total of 63 parentage confirmed paternity cases contained at least one mismatched locus have been collected.The mutations were subsequently evaluated using Verogen’s MPSForenSeqTM DNASignature Kit and a microsatellite instability(MSI)detection kit.The result showed 98.41%(62/63)of the cases had no additional autosomal mutations even when the number of A-STRs increased to 27.As for the sex chromosomes,about 11.11%(7/63)of the cases exhibited either X-STR or Y-STR mutations.D2S1338,FGAand Penta Ewere the most frequent altered STRs,which suggested they might be the mutation hotspots.In addition,a male with sex chromosome abnormality was observed accidently,whose genotype might be 47,XXY,rather than MSI.Nearly 56.90%of the STR loci possessed isoalleles,which might result in higher STR polymorphisms.No Mendelian incompatibility was detected among the SNP markers,which indicated that SNP was a more reliable genetic marker in the genetic-inconsistent paternity cases.

A novel mutation in the SRY gene of a Chinese 46,XY female patient with unilateral mixed germ cell tumor

Objective To determine the nosogenetic factors of a 46,XY female with primary amenorrhea and unilateral mixed germ cell tumor.Methods Eight genes associated with 46,XY gonadal dysgenesis were detected in the patient and her parents by target region captured-next generation sequencing.Results An insertion of a single nucleotide（adenine） at the coding site 230（c.230231insA） located in the high mobility group（HMG） domain of SRY was revealed,which led to a truncated protein（p.Lys77 fsX 27）. This mutation was at position 2655414 of the Y chromosome, supported with 127 unique mapped reads, however, this mutation was not found in the in-house dataset of 1 092 controls. Additionally, none of the candidate gene was detected in the patient’s parents, which indicated that it is a de novo mutation.Conclusion A novel SRY sporadic mutation due to a single nucleotide insertion at position 230（c.230231insA） was identified as the cause of the disease in this patient.Target region captured-next generation sequencing was found to be an effective method for the molecular genetic testing of 46,XY complete gonadal dysgenesis（46,XY CGD）.