Genome complexity of harmful microalgae

Various approaches can be implemented to identify a gene responsible for a phenotype of interest due to mutagenesis. Current next generation sequencing (NGS) technology allows whole genome sequencing of a mutant and accelerates the identification of mutation-induced polymorphisms in mutagenised organisms. In the model legume Lotus japonicus ecotype Miyakojima (MG-20), an abscisic acid (ABA) insensitive mutant called Beyma was previously isolated by ethyl methanesulphonate (EMS) mutagenesis and originally identified as a heterozygous dominant mutation. Beyma is slow-growing, wilty and incapable of regulating stomatal opening. A wild type segregant of the Beyma mutant (WTS) was also isolated from a self-generation of heterozygous Beyma mutants. ABA plays numerous roles in plant growth and development as well as morphogenetic responses including nodulation in legumes. Yet, there is a lack of ABA studies in legumes. Therefore, the Beyma mutant in L. japonicus allows a wide range of studies that will provide in-depth information of ABA signaling in nodulation as well as stress responses in legumes. This project presents an attempt to identify a causal gene in the ABA insensitive Beyma using the NGS technology. Tissue from a homozygous Beyma mutant, WTS and MG-20 wild type (WT) plants was subjected to the whole genome sequencing, generating about 300 million paired end of short-sequence reads. The Kazusa MG-20 genome was used as reference for read mapping. Single nucleotide polymorphisms (SNPs) were called based on mutations in the Beyma and WTS genomes as compared to the re-sequenced MG-20 genome. As a preliminary study, three procedures of read mapping and variant calling were performed to undertake a genomic comparative analysis and identify the causal gene. Sequencing of single genomes of the three plants showed a mutation occurred in every 208 kb (WTS) and 202 kb (Beyma) with a bias mutation of G/C-to-A/T changes at low percentage. Most mutations were intergenic. The mutation spectrum of the genomes was comparable in their individual chromosomes but each mutated genome has unique alterations, which are useful to identify causal mutations for their phenotypic changes. A total of 59 SNPs were identified as potential putative causal Beyma mutations, which were located in various annotated genes in the MG-20 genome. Verification of these mutations could not be done due to time constraint but will be performed in future. A candidate gene ii approach was also carried out by selecting ABA-related genes based on their roles in ABA biosynthesis to signalling, directly or indirectly. Mutations were found in loci of ABA INSENSITIVE (ABI) 1, ABI2, HAB1, HAB2, ABI3, ABI4, and ABSCISIC ACID 8'-HYDROXYLASE 2 in both mutant genomes or only in the WTS genome. Unique mutations also occurred in EARLY RESPONSIVE TO DEHYDRATION 7 and ABSCISIC ACID 8'-HYDROXYLASE 1/ P450 CYP707A1 genes, which were predicted to be impaired in their downstream regions. Although the candidates were not affected in the essential region of the genes, the candidate gene approach has eliminated all the candidates as the putatively causal Beyma gene. In order to intensify the identification of the causal Beyma gene, re-sequencing of the Beyma and WTS genomes was performed on pooled DNA. In this analysis, the presence of mutations was more frequent in both mutagenised genomes (~18-35% increase), resulting in higher rate of base changes and demonstrated that pooled DNA sequencing increased the mutation frequency. In addition, 69 unique Beyma SNPs were predicted to cause nonsynonymous changes and will be verified in future study. Nevertheless, a mutation (locus named chr3.CM0451.1060.r2.d) was found in both batches of sequencing. It was a C-to-T mutation, which changed glutamic acid to lysine in an F-box family gene. This gene could be the Beyma gene but it requires verification. In conjunction with the genome sequence analysis, other analyses were also done to prepare plant materials for sequencing and future verification. Plants were subjected to ABA treatment on seed germination and root development to select good mutant lines and WTS plants. Outcross between Beyma and L. japoncus ecotype Gifu was also performed for the segregation analysis of the putative causal SNPs in the F2 plants carrying homozygous WT alleles. This project highlighted the overall molecular changes produced in the whole genome of MG-20 mutants due to EMS mutagenesis. In future, the identification of the causal Beyma gene will possibly show a novel gene involved in ABA sensitivity in legume systems. In addition, it should be of great interest for researchers in forward genetics in legume studies.

A number of next-generation sequencing (NGS) technologies such as Roche/454, Illumina and AB SOLiD have recently become available. These technologies are capable of generating hundreds of thousands or tens of millions of short DNA sequence reads at a relatively low cost. These NGS technologies, now referred as second-generation sequencing (SGS) technologies, are being utilized for de novo sequencing, genome re-sequencing, and whole genome and transcriptome analysis. Now, new generation of sequencers, based on the 'next-next' or third-generation sequencing (TGS) technologies like the Single-Molecule Real-Time (SMRT™) Sequencer, Heliscope™ Single Molecule Sequencer, and the Ion Personal Genome Machine™ are becoming available that are capable of generating longer sequence reads in a shorter time and at even lower costs per instrument run. Ever declining sequencing costs and increased data output and sample throughput for NGS and TGS sequencing technologies enable the plant genomics and breeding community to undertake genotyping-by-sequencing (GBS). Data analysis, storage and management of large-scale second or TGS projects, however, are essential. This article provides an overview of different sequencing technologies with an emphasis on forthcoming TGS technologies and bioinformatics tools required for the latest evolution of DNA sequencing platforms.

High‐throughput genotyping in estimating genetic resources and detecting pathogens in aquaculture

Collection and storage of HLA NGS genotyping data for the 17th International HLA and Immunogenetics Workshop

Towards standardization of the description and publication of next‐generation sequencing datasets of fungal communities

Validation of NGS for mitochondrial DNA casework at the FBI Laboratory

Objective To investigate the value of next-generation sequencing (NGS) technique for genetic analysis of spontaneous abortion. Methods From January to June 2017, 154 patients who visited the First Affiliated Hospital of Zhengzhou University for spontaneous abortion were enrolled. All abortion tissue samples were analyzed by both NGS combined with short tandem repeat (STR) and single nucleotide polymorphism array (SNP-array). Results of the two methods were compared by Chi-square or Fisher's exact test. Results (1) Chromosomal abnormalities were detected in 109 of the 154 cases (70.7%), including 52 (47.7%) of numerical chromosomal abnormalities, 49 (45.0%) of structural chromosomal abnormalities, six (5.5%) of mosaicism, and two (1.8%) of uniparental disomy (UPD). In those 52 cases of numerical chromosome abnormalities, there were 45 of chromosome aneuploidy and seven of polyploidy. The top three numerical chromosomal abnormalities were 45,X (27.0%, 14/52), trisomy 22 (9.6%, 5/52) and trisomy 16 (7.7%, 4/52). Forty-nine structural abnormality cases carried 67 copy number variations (CNV), including 13 pathogenic CNV (pCNV, 19.4%), 24 variants of unknown clinical significance (35.8%) and 30 benign CNV (44.8%). In those 13 pCNVs, two were responsible for microdeletion and microduplication syndromes. (2) SNP-array was successful in 152 cases, but failed in two (1.3%) due to genomic DNA <200 ng. However, NGS technology was successful in all 154 cases and identified chromosomal abnormalities in the two cases that SNP-array had failed. No statistically significant difference was shown in the detection rate of chromosomal abnormalities between SNP-array and NGS technology [70.4% (107/152) vs 67.5% (104/154), χ2=0.293, P=0.588]. (3) No significant difference in the detection of chromosome aneuploidy (six cases in each group, 3.9% vs 3.9%) and mosaicism (45 cases in each group, 29.2% vs 29.6%) was found between NGS technology and SNP-array. Three cases of polyploidy (69, XXX) and two of UPD were identified by SNP-array, but not by NGS. When combined with STR, NGS was able to detect all three cases of polyploidy (69, XXX). (4) Forty-seven structural abnormality cases detected by SNP-array carried 53 CNVs, and 49 detected by NGS carried 67 CNVs. (5) NGS detected ten, three and one more CNVs than SNP-array did when the genome lengths were 100-<500, 500-<1 000 and ≥1 000 kb, respectively. Conclusions NGS can be used to detect chromosomal aneuploidy and mosaicism that can be identified by SNP-array with fewer limitations on total amount of genome. Moreover, CNVs that fail to be identified by SNP-array can also be detected by NGS. When combined with STR, NGS can effectively detect chromosomal polyploidy. Therefore, NGS could be a potential genetic analysis method for spontaneous abortion and of importance for genetic counseling. Key words: Abortion, spontaneous; High-throughput nucleotide sequencing; Chromosome aberrations; Polymorphism, single nucleotide

Background: The recent introduction of clinical Next Generation Sequencing (NGS) technologies has transformed the ability of health care providers to provide personalized hereditary breast cancer risk assessments to patients and their family members. However, along with the plethora of data derived from NGS technologies come challenges regarding how to interpret this data in a clinically meaning way to guide medical management decisions. Methods: A retrospective analysis was performed on a population of 242 patients with a personal or family history of breast and/or ovarian cancer and completed NGS genetic testing between March 2012 and May 2014 at Ambry Genetics. Data points included age, gender, primary diagnosis, type of NGS genetic test and result. Patients with prior genetic testing were not excluded from the analysis. Results: Of the 242 patients who completed NGS genetic testing 199 (82%) had a personal history of cancer and 43 (18%) had a family history of cancer. Ten (5%) clinically significant deleterious mutations were found in the affected population; 3 MUTYH mutations were consider not clinically significant and excluded from the analysis. Four (40%) were CHEK2 mutations, 2(20%) BRCA1 mutations, 1(10%) NBN mutation, 1(10%) ATM mutation, 1(10%) MSH6 mutation and 1(10%) PTEN mutation. One patient with a history of ovarian cancer had two deleterious mutations in the BRCA1 and NBN genes, and three affected patients had a deleterious mutation in addition to a variant of uncertain significance (VUS); a patient with synchronous breast and ovarian cancers had a MSH6 mutation and a BRCA2 VUS, a patient with breast cancer had a CHEK2 mutation and a CHEK2 VUS, and a patient with breast cancer had a CHEK2 mutation and a MSH6 VUS. A total of 37 (19%) VUS were found in the affected population, including one patient with a history of breast cancer and a VUS in both ATM and MRE11A. A single (2%) clinically significant BRCA1 deleterious mutation and 6(14%) VUS were detected in the unaffected population. Conclusions: NGS technologies allow for health care providers to complete more comprehensive hereditary breast cancer risk assessments for patients and their families. However, the high VUS rate (14-19%) and the possibility for multiple mutations and/or variants in a single patient create unique challenges when interpreting NGS genetic test results. Furthermore, the paucity of prospective studies and consensus guidelines limits the ability of the health care provider to interpret NGS genetic test results in a clinically meaningful way to guide patient management. This is highlighted by the case example above regarding the patient with synchronous breast and ovarian cancers who was found to have a MSH6 mutation and a BRCA2 VUS. Given that the patient does not meet Amsterdam criteria, the MSH6 mutations would have been missed had it not been for the NGS technology utilized for this patient. However, challenges still exist about how to interpret this data for both the patient and her family in a clinically meaningful way given that the family does not look like a classic Lynch Syndrome family. Citation Format: Barbara Hamlington, Scot Sedlacek, Lucy Langer, Brittney Goetsch, Katie Lemas, Sami Diab. Utilizing next generation sequencing technologies for hereditary breast cancer risk assessments in a private oncology practice [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-12-08.

Thanks to recent advances in next-generation sequencing (NGS) technology, diverse livestock species have been dissected at the genome-wide sequence level. As for cattle, there are currently four Korean indigenous breeds registered with the Domestic Animal Diversity Information System of the Food and Agricultural Organization of the United Nations: Hanwoo, Chikso, Heugu, and Jeju Heugu. These native genetic resources were recently whole-genome resequenced using various NGS technologies, providing enormous single nucleotide polymorphism information across the genomes. The NGS application further provided biological such that Korean native cattle are genetically distant from some cattle breeds of European origins. In addition, the NGS technology was successfully applied to detect structural variations, particularly copy number variations that were usually difficult to identify at the genome-wide level with reasonable accuracy. Despite the success, those recent studies also showed an inherent limitation in sequencing only a representative individual of each breed. To elucidate the biological implications of the sequenced data, further confirmatory studies should be followed by sequencing or validating the population of each breed. Because NGS sequencing prices have consistently dropped, various population genomic theories can now be applied to the sequencing data obtained from the population of each breed of interest. There are still few such population studies available for the Korean native cattle breeds, but this situation will soon be improved with the recent initiative for NGS sequencing of diverse native livestock resources, including the Korean native cattle breeds.

P842: Unified comprehensive analysis of NGS and optical genome mapping data for constitutional applications using Bionano VIA software

The growing accessibility to genomic resources using next-generation sequencing (NGS) technologies has revolutionized the application of molecular genetic tools to ecology and evolutionary studies in non-model organisms. Here we present the case study of the European hake (Merluccius merluccius), one of the most important demersal resources of European fisheries. Two sequencing platforms, the Roche 454 FLX (454) and the Illumina Genome Analyzer (GAII), were used for Single Nucleotide Polymorphisms (SNPs) discovery in the hake muscle transcriptome. De novo transcriptome assembly into unique contigs, annotation, and in silico SNP detection were carried out in parallel for 454 and GAII sequence data. High-throughput genotyping using the Illumina GoldenGate assay was performed for validating 1,536 putative SNPs. Validation results were analysed to compare the performances of 454 and GAII methods and to evaluate the role of several variables (e.g. sequencing depth, intron-exon structure, sequence quality and annotation). Despite well-known differences in sequence length and throughput, the two approaches showed similar assay conversion rates (approximately 43%) and percentages of polymorphic loci (67.5% and 63.3% for GAII and 454, respectively). Both NGS platforms therefore demonstrated to be suitable for large scale identification of SNPs in transcribed regions of non-model species, although the lack of a reference genome profoundly affects the genotyping success rate. The overall efficiency, however, can be improved using strict quality and filtering criteria for SNP selection (sequence quality, intron-exon structure, target region score).

Thalassemia is a severe hereditary blood disorder that poses a significant threat to human health and leads to mortality and disability. It is one of the most prevalent monogenic diseases worldwide. The aim of this study was to analyze the molecular epidemiological data of individuals of childbearing age from the Han ethnic group with thalassemia in Southwest China and to explore the application of next-generation sequencing (NGS) technology in screening thalassemia carriers. The participants were Han males and females of childbearing age who sought medical advice at the West China Second University Hospital, Sichuan University from June 2022 to June 2023. We detected α- and β-thalassemia mutations using full-length capture of the thalassemia genes and NGS technology. In a cohort of 1,093 participants, 130 thalassemia carriers were identified, with an overall detection rate of 11.89% (130/1,093). Among these, 0.91% (10/1,093) had mutations that could not be detected using traditional PCR techniques. The proportions of carriers with α-, β-, and α-complexed β-thalassemia gene mutations were 7.68% (84/1,093), 3.93% (43/1,093), and 0.27% (3/1,093), respectively. We identified a novel HBA2 c.166del variant that has not been previously reported. Using NGS technology, we found that the mutation-carrying rate of thalassemia genes was 11.89% in the Han population of childbearing age in Southwest China. Compared with the results of traditional PCR techniques, NGS detected an additional 0.91% (10/1,093) rare genetic variants. NGS technology should be utilized as the primary screening method for thalassemia carriers among Han nationality people of childbearing age in Southwest China.

Sensitive detection of internal tandem duplication (ITD) mutation of FLT3 is very important in acute myeloid leukemia. To increase detection sensitivity of FLT3-ITD, we developed new detection algorithm using next generation sequencing (NGS) data. We validated results using nested polymerase chain reaction (PCR) methods. We compared results of NGS data, nested PCR and conventional PCR methods. First, using whole exome sequencing data of 83 AML patients, we applied calling algorithm for FLT3-ITD. Briefly, to detect ITDs with NGS data, the reads are aligned to a reference sequence (UCSC hg19), with BWA which is a read aligner allowing soft-clipping. Some reads can be an indication of the occurrence of ITD and BWA aligns those reads as soft-clipped. Second, we deigned two types of primer for Nested PCR. The first primer was targeted wildly for between exon14 and exon15 of FLT3 gene. Nested PCR primer was deigned to target previously reported regions which are frequently occurred ITD mutation. PCR reactions of two steps were performed using the PCR primers sequentially. In these 83 patients, FLT3-ITD was positive only in 7 patients when tested by conventional PCR methods. When NGS detection method was applied, this resulted in positive FLT3-ITD in 11 patients (11/83, 13%). When validation was performed using nested PCR, FLT3-ITD was confirmed in all of 11 patients. Nested PCR detected additional 4 patient positive for FLT3-ITD in this population. For 68 patients, FLT3-ITD was negative by both NGS and nested PCR method. Overall, NGS method improved sensitivity of FLT3-ITD detection by 57% in this population. And the concordance rate of NGS method and nested PCR was 95.2% (79/83). Then we investigated clinical significance of sensitive FLT3-ITD detection. For this, we performed nested PCR and conventional PCR at the same time in 238 AML patients to detect FLT3-ITD. Positive rate for FLT3-ITD was 20% (48/238) and 10% (24/238) by nested PCR and conventional PCR respectively. When survival analysis was performed, among patients with negative FLT3-ITD result by conventional PCR, patients who showed positive for FLT3-ITD by nested PCR had shorter overall survival compared to those who showed negative for FLT3-ITD by nested PCR. (p = 0.03). This implies that sensitive FLT3-ITD detection using nested PCR is clinically meaningful. Diagnosis of FLT3-ITD is very important genetic factor, leading a therapeutic direction for AML patient. Here we report that we have developed alternative more sensitive detection methods for FLT3-ITD based on nested PCR and NGS. Sensitive detection of FLT3-ITD was clinically meaningful, suggesting that these methods should be incorporated in a future clinical practice. Also, we want to note that, NGS method is capable of quantifying FLT3-ITD size and amount in AML patients. Citation Format: Daeyoon Kim, Yoojin Hong, Youngil Koh, Sung-Soo Yoon, Choong-Hyun Sun, Kwang-Sung Ahn, Seungmook Lee, Hongseok Yun, Suyeon Lee. Improved sensitive detection method of FLT3 (FMS-like tyrosine kinase) internal tandem duplication (ITD) mutation using next-generation sequencing technology and nested PCR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3186.

Background. High incidence and mortality rate tumours present a more favourable prognosis if diagnosed in early stages (Jemal A. et al, 2011). Screening programs based on effective radiological techniques (e.g. mammography or low dose spiral CT) promise a mortality reduction, but their application on large scale suffers of disadvantages such as the use of ionizing radiation, overdiagnosis risk and high costs. Thus, the identification of molecular markers for cancer early diagnosis, especially if detectable through a simple non-invasive blood test, would provide powerful tools for the improvement of screening programs. Recently, a novel class of small non-coding RNAs, namely microRNAs, has been identified. MiRNA are extremely stable in biological fluids and their levels are different in malignant vs. control sera, making them a promising class of biological markers for early diagnosis. Indeed, studies from our and other labs led to the identification of “miRNA signatures” able to predict the presence of tumours even within a population of asymptomatic high-risk individuals (Bianchi F. et al., 2011; Boeri M. et al., 2011). However, methods used in these studies rely on QPCR approaches, which require previous knowledge on the molecules to be investigated. Indeed, the real complexity of circulating RNAs is still obscure, comprising other classes of molecules not yet investigated that could behave as novel biomarkers. The recent development of high-throughput sequencing technologies (Next Generation Sequencing - NGS) provided instruments to reveal the complexity of nucleic acids, but a complete protocol for the identification of circulating RNAs has not been established nor compared with current available approaches (i.e. QPCR). Aim: Our aim is to develop a simultaneous and comparative protocol for serum miRNA analysis, which encompasses both high-throughput QPCR and sequencing analysis in order to reveal the complexity of circulating small-RNAs. Methods: Total RNA has been isolated from the serum of healthy donors and analysed simultaneously by low-density QPCR (TaqMan Low Density Array, Life Technologies) and by NGS (Illumina). Results: A step-wise protocol to simultaneously analyze non-coding RNAs using NGS technology (Illumina) and high-throughput QPCR has been developed. The protocol has been optimized to allow both analyses using even limited amount of serum (up to 1mL). Both platforms resulted as highly efficient and quantitative, although NGS manages to score many molecules and RNA species that could not be analysed by current QPCR platforms, thus revealing a major complexity of circulating non-coding RNAs. Conclusion: We proposed that the combined use of NGS and QPCR platforms would allow a wider and more detailed analysis of circulating RNAs, expanding our ability to fish out robust and efficient molecular markers for early detection of cancer. Citation Format: Francesca Montani, Rose Mary Carletti, Matteo J. Marzi, Gabriele Bucci, Francesco Nicassio, Pier Paolo Di Fiore. Identification of serum circulating non-coding RNAs as diagnostic markers by Next Generation Sequencing (NGS) and low-density array. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5280. doi:10.1158/1538-7445.AM2013-5280

Brucellosis is a common zoonotic disease in Iran. Antimicrobial-resistant (AMR) Brucella isolates have been reported from different developing countries, posing an imminent health hazard. The objective of this study was to evaluate AMR and virulence-associated factors in Brucella isolates recovered from humans and animals in different regions of Iran using classical phenotyping and next generation sequencing (NGS) technology. Our findings revealed that B. melitensis is the most common species in bovines, small ruminants and camels. B. abortus was isolated only from one human case. Probable intermediate or resistant phenotype patterns for rifampicin, trimethoprim-sulfamethoxazole, ampicillin-sulbactam and colistin were found. Whole genome sequencing (WGS) identified mprF, bepG, bepF, bepC, bepE, and bepD in all isolates but failed to determine other classical AMR genes. Forty-three genes associated with five virulence factors were identified in the genomes of all Brucella isolates, and no difference in the distribution of virulence-associated genes was found. Of them, 27 genes were associated with lipopolysaccharide (LPS), 12 genes were related to a type IV secretion system (virB1-B12), two were associated with the toll-interleukin-1 receptor (TIR) domain-containing proteins (btpA, btpB), one gene encoded the Rab2 interacting conserved protein A (ricA) and one was associated with the production of cyclic β-1,2 glucans (cgs). This is the first investigation reporting the molecular-based AMR and virulence factors in brucellae isolated from different animal hosts and humans in Iran. Iranian B. abortus and B. melitensis isolates are still in vitro susceptible to the majority of antibiotics used for the treatment of human brucellosis. WGS failed to determine classical AMR genes and no difference was found in the distribution of virulence-associated genes in all isolates. Still, the absence of classical AMR genes in genomes of resistant strains is puzzling, and investigation of phenotypic resistance mechanisms at the proteomic and transcriptomic levels is needed.