Single-molecule Real-time DNA Sequencing Research Articles

Identification of 43 novel HLA alleles by PacBio single molecule real-time sequencing in haematopoietic cell donors.

A total of 43 novel HLA alleles detected in haematopoietic cell donors using single molecule real-time DNA sequencing.

Secondary Metabolite Analysis of Phomopsis sp. LGT-5 Based on the Dual Guidance of Whole-Genome Sequencing and HPLC-Q-ToF-MS/MS.

Microorganisms produce a wealth of structurally diverse specialized metabolites with a remarkable range of biological activities. The Phomopsis sp. LGT-5 was obtained through tissue block and repeatedly crossed methods from Tripterygium wilfordii Hook. F. The antibacterial experiments of LGT-5 showed that it has high inhibitory activity against Staphylococcus aureus and Pseudomonas aeruginosa, and moderate inhibitory activity against Candida albicans. To research the generation of the antibacterial phenomenon of LGT-5 and provide support for further research and application, the whole genome sequencing (WGS) of LGT-5 was obtained by single-molecule real-time DNA sequencing platform Pacific Biosciences (PacBio) sequencing and Illumina paired-end sequencing. The final assembled LGT-5 genome is 54.79 Mb with a contig N50 of 290.07 kb; in addition, its secondary metabolites were detected through HPLC-Q-ToF-MS/MS. By comparing its MS/MS data, the secondary metabolites were analyzed based on visual network maps obtained on the Global Natural Products Social Molecular Networking (GNPS). The analysis results showed that the secondary metabolites of LGT-5 were triterpenes and various cyclic dipeptides.

Analysis of secondary metabolite gene clusters and chitin biosynthesis pathways of Monascus purpureus with high production of pigment and citrinin based on whole-genome sequencing.

Monascus is a filamentous fungus that is widely used for producing Monascus pigments in the food industry in Southeast Asia. While the development of bioinformatics has helped elucidate the molecular mechanism underlying metabolic engineering of secondary metabolite biosynthesis, the biological information on the metabolic engineering of the morphology of Monascus remains unclear. In this study, the whole genome of M. purpureus CSU-M183 strain was sequenced using combined single-molecule real-time DNA sequencing and next-generation sequencing platforms. The length of the genome assembly was 23.75 Mb in size with a GC content of 49.13%, 69 genomic contigs and encoded 7305 putative predicted genes. In addition, we identified the secondary metabolite biosynthetic gene clusters and the chitin synthesis pathway in the genome of the high pigment-producing M. purpureus CSU-M183 strain. Furthermore, it is shown that the expression levels of most Monascus pigment and citrinin clusters located genes were significantly enhanced via atmospheric room temperature plasma mutagenesis. The results provide a basis for understanding the secondary metabolite biosynthesis, and constructing the metabolic engineering of the morphology of Monascus.

Whole-genome sequence of the planarian Dugesia japonica combining Illumina and PacBio data.

Advances in stem cell biology have posed the challenges in revealing the mechanistic themes underlying whole tissues and organs formation during regeneration. The planarian Dugesia japonica is an ideal model organism for the study of regeneration and stem cell biology. However, the genome resources for this species are still limited. Here, we combined single-molecule real-time DNA sequencing platform Pacific Biosciences (PacBio) sequencing, Illumina paired-end sequencing and 10× Genomics linked reads data to obtain the whole-genome sequence of the planarian D. japonica. The final assembled D. japonica genome is 1.13G with contig N50 of 248.44kb, and scaffold N50 of 652.52kb. Repeat elements account for 64.92% of the genome, and 12,031 protein coding genes were annotated, of which 10,114 genes had at least one functional annotation, representing 84.07% of the total genes. We present a highly contiguous genome assembly of D. japonica. The D. japonica genome assembly, together with gene annotation and transcriptome data provide a valuable resource to investigate molecular mechanism of planarian and stem cell research.

Effects of storage methods on the microbial community and quality of Sichuan smoked bacon

This study examined the storage of smoked bacon. Gas chromatography-mass spectrometry (GC–MS) and single-molecule real-time DNA sequencing technology (SMRT) were used to quantitate volatile constituents and microbial communities. The results indicated that the quality of bacon and its volatile constituents at room temperature are higher than those stored at 4 °C (P < 0.05). The bacterial abundance of smoked bacon in the H group that were hung from a beam at room temperature was higher than those of the L group, which were packaged under vacuum conditions and stored at 4 °C. The fungal diversity was lower in the H group than in the L group. Staphylococcus equorum was the most prominent bacterial species in the two groups, while both the fungal species Candida zeylanoides and Debaryomyces prosopidis were in all groups of smoked bacon. Pearson correlation coefficients indicated that Staphylococcus equorum positively correlated with heptanal in both groups. Lactobacillus sakei was positively (P < 0.01) related to heptanal in refrigeration, and Debaryomyces prosopidis positively correlated with hexanal in both groups. This study should help to improve the quality of meat by mediating microbial communities in Sichuan smoked bacon.

The Aryne Phosphate Reaction.

Condensed phosphates are a critically important class of molecules in biochemistry. Non‐natural analogues are important for various applications, such as single‐molecule real‐time DNA sequencing. Often, such analogues contain more than three phosphate units in their oligophosphate chain. Consequently, investigations into phosphate reactivity enabling new ways of phosphate functionalization and oligophosphorylation are essential. Here, we scrutinize the potential of phosphates to act as arynophiles, paving the way for follow‐up oligophosphorylation reactions. The aryne phosphate reaction is a powerful tool to—depending on the perspective—(oligo)phosphorylate arenes or arylate (oligo‐cyclo)phosphates. Based on Kobayashi‐type o‐silylaryltriflates, the aryne phosphate reaction enables rapid entry into a broad spectrum of arylated products, like monophosphates, diphosphates, phosphodiesters and polyphosphates. The synthetic potential of these new transformations is demonstrated by efficient syntheses of nucleotide analogues and an unprecedented one‐flask octaphosphorylation.

Single Molecule Real-Time Sequencing of the M Protein (SMaRT M-Seq): Toward Personalized Medicine Approaches in Monoclonal Gammopathies

IntroductionIn patients affected by monoclonal gammopathies, tumoral B cells or plasma cells secrete a monoclonal antibody (termed M protein), which can be used to track the presence of the tumor itself. Moreover, the M protein can directly cause potentially life-threatening organ damage, which is dictated by the specific, patient's unique clonal light and/or heavy chain, as in patients affected by immunoglobulin light chain (AL) amyloidosis. Yet, the current paradigm in the diagnosis and management of these conditions treats the M protein as a simple tumor biomarker to be identified/quantified. Patients' specific M protein sequences remain mostly undefined and molecular mechanisms underlying M-protein related clinical manifestations are largely obscure.MethodsBy combining the unbiased amplification of expressed immunoglobulin genes with long-read, single molecule real-time DNA sequencing and bioinformatics analyses, we have established a method to identify the full-length sequence of the variable region of expressed immunoglobulin genes and to rank the obtained sequences based on their relative abundance, thus enabling the identification of the full-length variable sequence of M protein genes from a high number of patients analysed in parallel.ResultsThe assay, which we termed Single Molecule Real-Time Sequencing of the M protein (SMaRT M-Seq), has undergone an extensive technical validation. Sequencing of contrived bone marrow samples generated through serial dilutions of plasma cell lines into control bone marrow, as well as sequencing of bona fide bone marrow samples from AL patients and comparison with gold-standard techniques of immunoglobulin gene sequencing showed: •100% sequence-accuracy at the individual base-pair level;•High repeatability (CV<0.8% for sequencing of pentaplicates) in defining the molecular clonal size (i.e. the fraction of total immunoglobulin sequences coinciding with the clonal sequence);•A high sensitivity in identifying clonal immunoglobulin sequences (10 -3 when employing low-coverage sequencing on multiple, pooled samples).Noteworthy, SMaRT M Seq was applied to a cohort of 86 consecutive patients with AL amyloidosis (17 κ and 69 λ; median BMPC infiltration 9%, IQR 6-13%; median dFLC 176 mg/L, IQR 75-370 mg/L), including cases with small clonal burden and M protein which was undetectable with conventional M protein studies. A full-length sequence of the variable region of the clonal light chain was obtained in all patients (median molecular clonal size of 88.3%, IQR: 70.7 - 93%). The most common κ germline genes were IGKV1-33 and IGKV4-01 (24% each of the 17 κ AL patients), and the most common λ germline genes were IGLV6-57 (26% of the 69 λ AL patients), IGLV2-14 (17%), IGLV3-01 (17%) and IGLV1-44 (10%). The most frequent λ and κ germline genes together (IGLV6-57, IGLV2-14, IGLV3-01, IGLV1-44, IGKV1-33 and IGKV4-01) accounted for 66% of all the clones. Germline gene usage correlated with selected clinical features.Sequence information was then exploited to improve mass spectrometry-based amyloid typing on fat pad aspirates and to enable the sensitive detection of clonotypic sequences using short-read DNA sequencing of the involved light chain isotype (up to 10 -7 dilution).ConclusionsWe have established SMaRT M-Seq as a novel valuable assay to reliably identify the full-length variable sequence of M proteins. SMaRT M-Seq has undergone extensive technical validation, showing high accuracy, repeatability and sensitivity. The latter is determined by the number of reads analyzed per sample. This is in turn dictated by the sequencing output of the employed sequencing platform, and by the number of pooled samples analyzed in a given sequencing round, thus proving to be scalable. Even when analyzing multiple samples on a sequencing platform with low sequencing output, the achieved sensitivity of SMaRT M-Seq significantly exceeds the requirements for the identification of clonal B cells/plasma cells in patients with AL amyloidosis.Sequencing disease-associated M proteins from large cohorts of patients has the potential to uncover molecular mechanisms of M protein-related clinical manifestations which have remained largely unexplored so far, and could enable approaches of personalized medicine for the sensitive detection of patients' specific M proteins at diagnosis and after anti-clonal therapy. DisclosuresMilani: Celgene: Other: Travel support; Janssen-Cilag: Honoraria. Fazio: Janseen: Honoraria. Petrucci: GSK: Honoraria, Other: Advisory Board; Amgen: Honoraria, Other: Advisory Board; Takeda: Honoraria, Other: Advisory Board; BMS: Honoraria, Other: Advisory Board; Janssen-Cilag: Honoraria, Other: Advisory Board; Celgene: Honoraria, Other: Advisory Board; Karyopharm: Honoraria, Other: Advisory Board. Palladini: Pfizer: Honoraria; Siemens: Honoraria; Janssen Global Services: Honoraria, Other: advisory board fees. Nuvolone: Janssen-Cilag: Honoraria; Oncopeptides, Inc.: Research Funding.

The volatile profiles and microbiota structures of the wheat Qus used as traditional fermentation starters of Chinese rice wine from Shaoxing region

Wheat Qu is used as a fermentation starter in Chinese rice wine brewing and plays important roles on the quality and flavor of the product. The aim of this study was to thoroughly investigate and compare ten traditional wheat Qus collected from different Chinese rice wine manufacturers in Shaoxing region in terms of their hydrolytic enzyme activities, volatile profiles and microbiota structures. Significant variation in the enzymatic activities (glucoamylase, amylase and protease) and volatile components were observed among the samples. Single molecule real-time DNA sequencing technology (SMRT) and culture dependent approach were combined for the first time to investigate the microbial communities in traditional wheat Qus. SMRT indicated that 4 bacterial genera, including Saccharopolyspora, Staphylococcus, Bacillus and Pantoea, and 5 fungal genera, including Aspergillus, Wickerhamomyces, Candida, Alternaria and Fusarium, dominated the wheat Qus and were detected in all ten samples. On the other hand, Staphylococcus gallinarum and Lichtheimia ramose were found to be the most abundant bacterial and fungal species by culture dependent method. Moreover, the potential correlations between microorganisms and volatiles of wheat Qus were also explored using partial least squares regression. Finally, this study provided detailed insight into the microbial communities and volatile profiles presented in wheat Qu.

Structural and functional diversity among Type III restriction-modification systems that confer host DNA protection via methylation of the N4 atom of cytosine.

We report a new subgroup of Type III Restriction-Modification systems that use m4C methylation for host protection. Recognition specificities for six such systems, each recognizing a novel motif, have been determined using single molecule real-time DNA sequencing. In contrast to all previously characterized Type III systems which modify adenine to m6A, protective methylation of the host genome in these new systems is achieved by the N4-methylation of a cytosine base in one strand of an asymmetric 4 to 6 base pair recognition motif. Type III systems are heterotrimeric enzyme complexes containing a single copy of an ATP-dependent restriction endonuclease-helicase (Res) and a dimeric DNA methyltransferase (Mod). The Type III Mods are beta-class amino-methyltransferases, examples of which form either N6-methyl adenine or N4-methyl cytosine in Type II RM systems. The Type III m4C Mod and Res proteins are diverged, suggesting ancient origin or that m4C modification has arisen from m6A MTases multiple times in diverged lineages. Two of the systems, from thermophilic organisms, required expression of both Mod and Res to efficiently methylate an E. coli host, unlike previous findings that Mod alone is proficient at modification, suggesting that the division of labor between protective methylation and restriction activities is atypical in these systems. Two of the characterized systems, and many homologous putative systems, appear to include a third protein; a conserved putative helicase/ATPase subunit of unknown function and located 5’ of the mod gene. The function of this additional ATPase is not yet known, but close homologs co-localize with the typical Mod and Res genes in hundreds of putative Type III systems. Our findings demonstrate a rich diversity within Type III RM systems.

4-Methylcytosine distribution follows the power function in Geobacter sulfurreducens genome

Single-molecule real-time DNA sequencing revealed that 4-methylcytosine (m4C) commonly exists in bacterial genomes. In this work, samples with different m4C methylation patterns were studied. Results reveal that m4C modification is a biochemical reaction with distance effect, and its distribution follows the power function in the positive, negative, and double strands of genomic DNA sequences of Geobacter sulfurreducens. Furthermore, the value of regression coefficient in the fitting formula for double strands was the sum of those in the fitting formulae for positive and negative strands. Meanwhile, the value of exponent coefficient was the average, implicating an interesting mathematical phenomenon about power function. Considering the potent role of m4C in gene expression and the present results being obtained from the same genomic DNA sequence, this work suggests that the patterns of m4C distribution may be served as a signal for G. sulfurreducens to rapidly identify the genes to respond to environmental stresses or signals. This study opens a new avenue to extend our knowledge about the modification mechanisms and the epigenetic information of m4C modification in prokaryotes.

Massively multiplex single-molecule oligonucleosome footprinting.

Our understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high-resolution sequence-agnostic imaging methods and sequence-resolved bulk biochemical techniques. To bridge the divide between these approaches, we present the single-molecule adenine methylated oligonucleosome sequencing assay (SAMOSA). SAMOSA is a high-throughput single-molecule sequencing method that combines adenine methyltransferase footprinting and single-molecule real-time DNA sequencing to natively and nondestructively measure nucleosome positions on individual chromatin fibres. SAMOSA data allows unbiased classification of single-molecular 'states' of nucleosome occupancy on individual chromatin fibres. We leverage this to estimate nucleosome regularity and spacing on single chromatin fibres genome-wide, at predicted transcription factor binding motifs, and across human epigenomic domains. Our analyses suggest that chromatin is comprised of both regular and irregular single-molecular oligonucleosome patterns that differ subtly in their relative abundance across epigenomic domains. This irregularity is particularly striking in constitutive heterochromatin, which has typically been viewed as a conformationally static entity. Our proof-of-concept study provides a powerful new methodology for studying nucleosome organization at a previously intractable resolution and offers up new avenues for modeling and visualizing higher order chromatin structure.

Single molecule real-time DNA sequencing of the full HLA-E gene for 212 reference cell lines.

We have developed a genotyping assay that produces fully phased, unambiguous HLA-E genotyping using Pacific Biosciences' single molecule real-time DNA sequencing. In total 212 cell lines were genotyped, including the panel of 107 established at the 10th International Histocompatibility Workshop. Our results matched the previously known HLA-E genotype in 94 (44.3%) cell lines, in all cases either improving or equalling previous genotyping resolution. Three (1.4%) cells had discrepant HLA-E genotyping data and 115 (54.2%) had no previous HLA-E data. The HLA-E genotypes for four (1.9%) cell lines resulted in a change of zygosity by identifying two distinct haplotypes. We discovered eight novel HLA-E alleles, extended the known reference sequence of seven and confirmed the existence of a further 10.

Combined use of single molecule real-time DNA sequencing technology and culture-dependent methods to analyze the functional microorganisms in inoculated raw wheat Qu

Inoculated raw wheat Qu (IRWQ), which can be produced with high efficiency and low cost while maintaining stable quality, is a new Qu that has been applied to Huangjiu brewing. In this study, single molecule real-time DNA sequencing technology and culture-dependent methods were combined for the first time to study the microbiota and the function of the principle microorganisms in IRWQ. The glucoamylase, amylase and protease contents of IRWQ were 1.17, 1.55 and 2.87-times greater, respectively, than those of traditional wheat Qu. Like traditional wheat Qu, the main volatile flavor compounds in IRWQ were alcohols; however, the esters content was much higher and the acids content was much lower. Single molecule real-time DNA sequencing technology identified 18 fungal species and 59 bacterial species in IRWQ. Then, 30 species were isolated by culture-dependent methods. These species represented about 80% of the total fungal microbiota and 35% of the total bacteria microbiota. Molds were the main contributors of glucoamylase, amylase and protease activities. Aspergillus flavus had the highest glucoamylase and protease activity. Bacillus subtilis and Bacillus amyloliquefaciens also produced high hydrolytic enzyme activities. Saccharomyces cerevisiae produced the largest amounts of aromatics compounds, alcohols, esters and acids. Aldehydes and ketones are mainly produced by molds.

Extending the sequences of HLA class I alleles without full-length genomic coverage using single molecule real-time DNA sequencing.

The assignment of an HLA allele name to a sequence requires a comparison between the generated target sequence and a reference sequence on the IPD-IMGT/HLA database. Absence of a full-length reference sequence can result in the inability of HLA typing software to accurately compare and assign the sequence. We sequenced the most frequently seen HLA class I alleles on the Anthony Nolan register present in the database with only a partial genomic sequence, with the aim of increasing the number of complete reference sequences. We successfully extended 95 full-length HLA class I sequences and identified 13 novel variants. Increasing the number of full-length HLA class I reference sequences in the database has aided accuracy of HLA analysis tools for all histocompatibility and immunogenetics laboratories.

Five Closed Salmonella enterica Genome Sequences from a 2017-2018 Multistrain, Multistate Kratom Outbreak.

We report here the closed genomes of Salmonella enterica strains from the 2017-2018 multistrain, multistate kratom outbreak using single-molecule real-time DNA sequencing. Four of the genomes consist of one circular chromosome, and the fifth has a circular chromosome and a single plasmid.

Large-scale and significant expression from pseudogenes in Sodalis glossinidius - a facultative bacterial endosymbiont.

The majority of bacterial genomes have high coding efficiencies, but there are some genomes of intracellular bacteria that have low gene density. The genome of the endosymbiont Sodalis glossinidius contains almost 50 % pseudogenes containing mutations that putatively silence them at the genomic level. We have applied multiple ‘omic’ strategies, combining Illumina and Pacific Biosciences Single-Molecule Real-Time DNA sequencing and annotation, stranded RNA sequencing and proteome analysis to better understand the transcriptional and translational landscape of Sodalis pseudogenes, and potential mechanisms for their control. Between 53 and 74 % of the Sodalis transcriptome remains active in cell-free culture. The mean sense transcription from coding domain sequences (CDSs) is four times greater than that from pseudogenes. Comparative genomic analysis of six Illumina-sequenced Sodalis isolates from different host Glossina species shows pseudogenes make up ~40 % of the 2729 genes in the core genome, suggesting that they are stable and/or that Sodalis is a recent introduction across the genus Glossina as a facultative symbiont. These data shed further light on the importance of transcriptional and translational control in deciphering host–microbe interactions. The combination of genomics, transcriptomics and proteomics gives a multidimensional perspective for studying prokaryotic genomes with a view to elucidating evolutionary adaptation to novel environmental niches.

Genome-wide systematic identification of methyltransferase recognition and modification patterns

Genome-wide analysis of DNA methylation patterns using single molecule real-time DNA sequencing has boosted the number of publicly available methylomes. However, there is a lack of tools coupling methylation patterns and the corresponding methyltransferase genes. Here we demonstrate a high-throughput method for coupling methyltransferases with their respective motifs, using automated cloning and analysing the methyltransferases in vectors carrying a strain-specific cassette containing all potential target sites. To validate the method, we analyse the genomes of the thermophile Moorella thermoacetica and the mesophile Acetobacterium woodii, two acetogenic bacteria having substantially modified genomes with 12 methylation motifs and a total of 23 methyltransferase genes. Using our method, we characterize the 23 methyltransferases, assign motifs to the respective enzymes and verify activity for 11 of the 12 motifs.

Methylome and Metabolome Analyses Reveal Adaptive Mechanisms in Geobacter sulfurreducens Grown on Different Terminal Electron Acceptors.

The Geobacter species evolved respiratory versatility to utilize a wide range of terminal electron acceptors. To explore this adaptive mechanism, Fe(III) citrate, hydrous ferric oxide, and fumarate were selected as electron acceptors, and the methylome and metabolome of Geobacter sulfurreducens PCA grown on each electron acceptor were investigated via third-generation, single-molecule real-time DNA sequencing and gas chromatography/time-of-flight mass spectrometry-based metabolomics, respectively. Results showed that the patterns of 4-methylcytosine (m4C) and 6-methyladenine (m6A) modification, the concentrations of fatty acids (e.g., caprylic acid, capric acid, and squalene), and the activity of antioxidant enzymes (e.g., superoxide dismutase, catalase, and glutathione reductase) were all varied in different electron acceptor cultures. Moreover, genes (e.g., GSU0466 and GSU1467) with low expression levels generally had high methylation levels. These findings suggest that m4C and m6A modifications, fatty acids, and antioxidant enzymes all play a role in the adaptation of G. sulfurreducens to diverse electron acceptors, and DNA methylation may be involved in the adaptation mainly via gene expression regulation.

HLA-DPB1*64:01N and DPB1*701:01 sequence extensions by single molecule real-time DNA sequencing.

Pacific Bioscience's SMRT sequencing was used to confirm and extend HLA-DPB1*64:01N and 701:01.

A draft genome assembly of the Chinese sillago (Sillago sinica), the first reference genome for Sillaginidae fishes.

BackgroundSillaginidae, also known as smelt-whitings, is a family of benthic coastal marine fishes in the Indo-West Pacific that have high ecological and economic importance. Many Sillaginidae species, including the Chinese sillago (Sillago sinica), have been recently described in China, providing valuable material to analyze genetic diversification of the family Sillaginidae. Here, we constructed a reference genome for the Chinese sillago, with the aim to set up a platform for comparative analysis of all species in this family.FindingsUsing the single-molecule real-time DNA sequencing platform Pacific Biosciences (PacBio) Sequel, we generated ∼27.3 Gb genomic DNA sequences for the Chinese sillago. We reconstructed a genome assembly of 534 Mb using a strategy that takes advantage of complementary strengths of two genome assembly programs, Canu and FALCON. The genome size was consistent with the estimated genome size based on k-mer analysis. The assembled genome consisted of 802 contigs with a contig N50 length of 2.6 Mb. We annotated 22,122 protein-coding genes in the Chinese sillago genomes using a de novo method as well as RNA sequencing data and homologies to other teleosts. According to the phylogenetic analysis using protein-coding genes, the Chinese sillago is closely related to Larimichthys crocea and Dicentrarchus labrax and diverged from their ancestor around 69.5–82.6 million years ago.ConclusionsUsing long reads generated with PacBio sequencing technology, we have built a draft genome assembly for the Chinese sillago, which is the first reference genome for Sillaginidae species. This genome assembly sets a stage for comparative analysis of the diversification and adaptation of fishes in Sillaginidae.