Single Molecular Sequencing Research Articles

A high-quality genome of the early diverging tychoplanktonic diatom Paralia guyana

The diatom Paralia guyana is a tychoplanktonic microalgal species that represents one of the early diverging diatoms. P. guyana can thrive in both planktonic and benthic habitats, making a significant contribution to the occurrence of red tide events. Although a dozen diatom genomes have been sequenced, the identity of the early diverging diatoms remains elusive. The understanding of the evolutionary clades and mechanisms of ecological adaptation in P. guyana is limited by the absence of a high-quality genome assembly. In this study, the first high-quality genome assembly for the early diverging diatom P. guyana was established using PacBio single molecular sequencing. The assembled genome has a size of 558.85 Mb, making it the largest diatom genome on record, with a contig N50 size of 26.06 Mb. A total of 27,121 protein-coding genes were predicted in the P. guyana genome, of which 22,904 predicted genes (84.45%) were functionally annotated. This data and analysis provide innovative genomic resources for tychoplanktonic microalgal species and shed light on the evolutionary origins of diatoms.

DNA 5-methylcytosine detection and methylation phasing using PacBio circular consensus sequencing

Long single-molecular sequencing technologies, such as PacBio circular consensus sequencing (CCS) and nanopore sequencing, are advantageous in detecting DNA 5-methylcytosine in CpGs (5mCpGs), especially in repetitive genomic regions. However, existing methods for detecting 5mCpGs using PacBio CCS are less accurate and robust. Here, we present ccsmeth, a deep-learning method to detect DNA 5mCpGs using CCS reads. We sequence polymerase-chain-reaction treated and M.SssI-methyltransferase treated DNA of one human sample using PacBio CCS for training ccsmeth. Using long (≥10 Kb) CCS reads, ccsmeth achieves 0.90 accuracy and 0.97 Area Under the Curve on 5mCpG detection at single-molecule resolution. At the genome-wide site level, ccsmeth achieves >0.90 correlations with bisulfite sequencing and nanopore sequencing using only 10× reads. Furthermore, we develop a Nextflow pipeline, ccsmethphase, to detect haplotype-aware methylation using CCS reads, and then sequence a Chinese family trio to validate it. ccsmeth and ccsmethphase can be robust and accurate tools for detecting DNA 5-methylcytosines.

Single-Molecule Approach to 16S rRNA for Vaginal Microbiome Signatures in Response to Metronidazole Treatment.

Bacterial vaginosis (BV) is the most common infection of the lower reproductive tract among women of reproductive age, characterized by a depletion of health-associated Lactobacillus and an overgrowth of anaerobes. Metronidazole has been recommended as a first-line therapy for treating BV for decades. Although most cases are cured by the treatment, recurrent infections of BV seriously affect women's reproductive health. Until now, limited information on the vaginal microbiota has been explored at the species level. Here, we adopted a single molecular sequencing approach for the 16S rRNA gene, named FLAST (full-length assembly sequencing technology), to analyze the human vaginal microbiota that improved species-level resolution for taxonomy and identified microbiota alterations in the vaginal tract in response to treatment with metronidazole. Appling high-throughput sequencing, we identified 96 and 189 novel full-length 16S rRNA gene sequences in Lactobacillus and Prevotella, respectively, which had not previously been reported in vaginal samples. Moreover, we found that Lactobacillus iners was significantly enriched in the cured group before metronidazole treatment, and that was maintained in a high frequency after the treatment, suggesting an important role for this species in response to metronidazole treatment. Our research also highlights the importance of the single-molecule paradigm for progressing the field of microbiology and applying these insights to better understand the dynamic microbiota during BV treatment. Subsequent novel treatment approaches should be proposed to improve BV treatment outcomes, optimize the vaginal microbiome, and reduce gynecological and obstetric sequelae. IMPORTANCE Bacterial vaginosis (BV) is a common infectious disease of the reproductive tract. Metronidazole treatment, as the first line of treatment, frequently fails at recovery of the microbiome. However, the precise types of Lactobacillus and other bacteria involved in BV remain unclear, and this has resulted in a failure to identify potential markers to predict clinic outcomes. In this study, we adopted a 16S rRNA gene full-length assembly sequencing technology for the taxonomy analysis and evaluation of vaginal microbiota before and after treatment with metronidazole. We additionally identified 96 and 189 novel 16S rRNA gene sequences in Lactobacillus and Prevotella species, respectively, in vaginal samples, which improves our understanding of the vaginal microbiota. Moreover, we found that the abundance of Lactobacillus iners and Prevotella bivia before treatment was associated with a lack of cure. These potential biomarkers will help to facilitate future studies aimed at improving BV treatment outcomes, optimize the vaginal microbiome, and reduce adverse sexual and reproductive outcomes.

KngMap: Sensitive and Fast Mapping Algorithm for Noisy Long Reads Based on the K-Mer Neighborhood Graph

With the rapid development of single molecular sequencing (SMS) technologies such as PacBio single-molecule real-time and Oxford Nanopore sequencing, the output read length is continuously increasing, which has dramatical potentials on cutting-edge genomic applications. Mapping these reads to a reference genome is often the most fundamental and computing-intensive step for downstream analysis. However, these long reads contain higher sequencing errors and could more frequently span the breakpoints of structural variants (SVs) than those of shorter reads, leading to many unaligned reads or reads that are partially aligned for most state-of-the-art mappers. As a result, these methods usually focus on producing local mapping results for the query read rather than obtaining the whole end-to-end alignment. We introduce kngMap, a novel k-mer neighborhood graph-based mapper that is specifically designed to align long noisy SMS reads to a reference sequence. By benchmarking exhaustive experiments on both simulated and real-life SMS datasets to assess the performance of kngMap with ten other popular SMS mapping tools (e.g., BLASR, BWA-MEM, and minimap2), we demonstrated that kngMap has higher sensitivity that can align more reads and bases to the reference genome; meanwhile, kngMap can produce consecutive alignments for the whole read and span different categories of SVs in the reads. kngMap is implemented in C++ and supports multi-threading; the source code of kngMap can be downloaded for free at: https://github.com/zhang134/kngMap for academic usage.

Large Differences in the Haptophyte Phaeocystis globosa Mitochondrial Genomes Driven by Repeat Amplifications.

The haptophyte Phaeocystis globosa is a well-known species for its pivotal role in global carbon and sulfur cycles and for its capability of forming harmful algal blooms (HABs) with serious ecological consequences. Its mitochondrial genome (mtDNA) sequence has been reported in 2014 but it remains incomplete due to its long repeat sequences. In this study, we constructed the first full-length mtDNA of P. globosa, which was a circular genome with a size of 43,585 bp by applying the PacBio single molecular sequencing method. The mtDNA of this P. globosa strain (CNS00066), which was isolated from the Beibu Gulf, China, encoded 19 protein-coding genes (PCGs), 25 tRNA genes, and two rRNA genes. It contained two large repeat regions of 6.7 kb and ∼14.0 kb in length, respectively. The combined length of these two repeat regions, which were missing from the previous mtDNA assembly, accounted for almost half of the entire mtDNA and represented the longest repeat region among all sequenced haptophyte mtDNAs. In this study, we tested the hypothesis that repeat unit amplification is a driving force for different mtDNA sizes. Comparative analysis of mtDNAs of five additional P. globosa strains (four strains obtained in this study, and one strain previously published) revealed that all six mtDNAs shared identical numbers of genes but with dramatically different repeat regions. A homologous repeat unit was identified but with hugely different numbers of copies in all P. globosa strains. Thus, repeat amplification may represent an important driving force of mtDNA evolution in P. globosa.

Alternative splicing analysis of wheat glutamine synthase genes

<p id="C3">As a key enzyme for nitrogen assimilation in plant, glutamine synthetase (TaGS) is encoded by 12 genes in common wheat (<italic>Triticum aestivum</italic> L.), namely <italic>TaGS1;1-6A/6B/6D</italic>, <italic>TaGS1;2-4A/4B/4D</italic>, <italic>TaGS1;3-4A/4B/4D</italic>, and <italic>TaGS2-2A/2B/2D</italic>. Transcripts of TaGS genes were obtained using the single molecular sequencing technology, and the results showed that <italic>TaGS1;1-6A</italic> had one alternative splicing (AS) version of transcripts and <italic>TaGS1;1-6B</italic> had two AS versions. Compared with the TaGS encoded by normal transcript, TaGS1;1-6A-1 lacked part of the Gln_synt_N domain, and TaGS1;1-6B-3 lacked the Gln_synth_gly_rich_ site domain; <italic>TaGS1;1-6B-4 </italic>was a new AS event, and encoded a truncated GS without Gln_synth_gly_rich_site and Gln_synth_ cat_dom domain. The relative expression level of TaGS alternative splicing transcripts of wheat cultivars with different nitrogen use efficiencies showed that the relative expression level of <italic>TaGS1;1-6A-1 </italic>in leaves of YM49 (N-efficient) was significantly higher than that of XN509 (N-inefficient), and increased with the increase of nitrogen supply. In roots, the relative expression of <italic>TaGS1;1-6A-1</italic> increased with the increase of nitrogen supply in YM49, but decreased in XN509. <italic>TaGS1;1-6B-3</italic> showed a similar expression trend in YM49 and XN509, and high concentration of NO₃^- promoted the expression of <italic>TaGS1;1-6B-3</italic> in leaves while inhibited in roots. High concentration of NH₄⁺ inhibited the expression of <italic>TaGS1;1-6B-3</italic> in leaves, but had little effect in roots. <italic>TaGS1;1-6B-4</italic> was mainly expressed under low nitrogen condition, and its expression in roots of YM49 was not affected by NH₄⁺ supply. Understanding the AS transcript versions of TaGS genes is helpful to illustrate the functions of TaGS isozymes in nitrogen metabolism of wheat.

Low-complexity and highly robust barcodes for error-rich single molecular sequencing.

DNA barcodes are frequently corrupted due to insertion, deletion, and substitution errors during DNA synthesis, amplification and sequencing, resulting in index hopping. In this paper, we propose a new DNA barcode construction scheme that combines a cyclic block code with a predetermined pseudo-random sequence bit by bit to form bit pairs, and then converts the bit pairs to bases, i.e., the DNA barcodes. Then, we present a barcode identification scheme for noisy sequencing reads, which uses a combination of cyclic shifting and traditional dynamic programming to mark the insertion and deletion positions, and then performs erasure-and-error-correction decoding on the corrupted codewords. Furthermore, we verify the identification error rate of barcodes for multiple errors and evaluate the reliability of the barcodes in DNA context. This method can be easily generalized for constructing long barcodes, which may be used in scenarios with serious errors. Simulation results show that the bit error rate after identifying insertions/deletions is greatly reduced using the combination of cyclic shift and dynamic programming compared to using dynamic programming only. It indicates that the proposed method can effectively improve the accuracy for estimating insertion/deletion errors. And the overall identification error rate of the proposed method is lower than when the probability of each base mutation is less than 0.1, which is the typical scenario in third-generation sequencing.

A survey on de novo assembly methods for single‐molecular sequencing

BackgroundThe single‐molecular sequencing (SMS) is under rapid development and generating increasingly long and accurate sequences. De novo assembly of genomes from SMS sequences is a critical step for many genomic studies. To scale well with the developing trends of SMS, many de novo assemblers for SMS have been released. These assembly workflows can be categorized into two different kinds: the correction‐and‐assembly strategy and the assembly‐and‐correction strategy, both of which are gaining more and more attentions.ResultsIn this article we make a discussion on the characteristics of errors in SMS sequences. We then review the currently widely applied de novo assemblers for SMS sequences. We also describe computational methods relevant to de novo assembly, including the alignment methods and the error correction methods. Benchmarks are provided to analyze their performance on different datasets and to provide use guides on applying the computation methods.ConclusionWe make a detailed review on the latest development of de novo assembly and some relevant algorithms for SMS, including their rationales, solutions and results. Besides, we provide use guides on the algorithms based on their benchmark results. Finally we conclude the review by giving some developing trends of third generation sequencing (TGS).

Characterization of Drought-Responsive Transcriptome During Seed Germination in Adzuki Bean (Vigna angularis L.) by PacBio SMRT and Illumina Sequencing.

The full-length single-molecular sequencing and short reads Illumina sequencing were combined to generate the transcripts of adzuki bean with high-quality. A total of 17,636 loci and 60,454 transcripts were detected in this study. To characterize the drought-responsive genes during seed germination in adzuki bean, two varieties, i.e., tolerant and sensitive to drought stress, were selected to conduct analysis of alternative splicing dynamics (AS) and differentially expressed genes (DEGs) by combining the newly assembled draft genome and public adzuki bean reference genome. AS analysis indicated that both the two varieties underwent a little more AS events under control conditions than under drought stress. Among the AS events, IR (intron retention) predominately accounted for 34.3%, whereas AD (alternative donor site) was the least frequent with 15.8%. Meanwhile, 562 long non-coding RNAs, 409 fusion genes and 1208 transcription factors were identified. Moreover, a total of 5,337 DEGs were identified in comparison of the two varieties with drought or control treatments. Notably, 82 DEGs were discovered in the two varieties under drought stress, which might be the candidate in regulation of seed germination to answer for different drought tolerance. The DEGs encoded proteins involved in primary or second metabolism, plant hormone signal transduction, transcript or translation processes, ubiquitin proteasome system, transcription factor, transporters, and so on. The results facilitate to increase the knowledge about the mechanism of drought tolerance during crop seed germination, and provide reference for the breeding of drought-tolerant adzuki bean.

PacBio single molecule long-read sequencing provides insight into the complexity and diversity of the Pinctada fucata martensii transcriptome

BackgroundThe pearl oyster Pinctada fucata martensii is an economically valuable shellfish for seawater pearl production, and production of pearls depends on its growth. To date, the molecular mechanisms of the growth of this species remain poorly understood. The transcriptome sequencing has been considered to understanding of the complexity of mechanisms of the growth of P. f. martensii. The recently released genome sequences of P. f. martensii, as well as emerging Pacific Bioscience (PacBio) single-molecular sequencing technologies, provide an opportunity to thoroughly investigate these molecular mechanisms.ResultsHerein, the full-length transcriptome was analysed by combining PacBio single-molecule long-read sequencing (PacBio sequencing) and Illumina sequencing. A total of 20.65 Gb of clean data were generated, including 574,561 circular consensus reads, among which 443,944 full-length non-chimeric (FLNC) sequences were identified. Through transcript clustering analysis of FLNC reads, 32,755 consensus isoforms were identified, including 32,095 high-quality consensus sequences. After removing redundant reads, 16,388 transcripts were obtained, and 641 fusion transcripts were derived by performing fusion transcript prediction of consensus sequences. Alternative splicing analysis of the 16,388 transcripts was performed after accounting for redundancy, and 9097 gene loci were detected, including 1607 new gene loci and 14,946 newly discovered transcripts. The original boundary of 11,235 genes on the chromosomes was corrected, 12,025 complete open reading frame sequences and 635 long non-coding RNAs (LncRNAs) were predicted, and functional annotation of 13,482 new transcripts was achieved. Two thousand three hundred eighteen alternative splicing events were detected. A total of 228 differentially expressed transcripts (DETs) were identified between the largest (L) and smallest (S) pearl oysters. Compared with the S, the L showed 99 and 129 significantly up-and down-regulated DETs, respectively. Six of these DETs were further confirmed by quantitative real-time RT-PCR (RT-qPCR) in independent experiment.ConclusionsOur results significantly improve existing gene models and genome annotations, optimise the genome structure, and in-depth understanding of the complexity and diversity of the differential growth patterns of P. f. martensii.

Progress in the application of single molecular sequencing in tumor diagnosis

Tumor development is usually related to the genetic mutation and abnormal expression of multiple genes. Comprehensive analysis of tumor genome, transcriptome and epigenetics is very important for the rapid identification of disease-specific gene clusters and modification sites. Previously, the next-generation sequencing technology was mainly used to explore the information of genomes, however, it cannot meet the requirement of mechanical researches due to several problems such as difficult sequence assembly and leak detection of the low abundance factors. Therefore, single molecule sequencing technology gradually emerged with its unique superiority. This paper reviews the research processes of single molecule sequencing technology in several human tumors, and prospecs its application in clinical diagnosis.

Complete genome sequence of novel isolate SYJ15 of Bacillus cereus group, a highly lethal pathogen isolated from Chinese soft shell turtle (Pelodiscus Sinensis).

SYJ15 is a highly pathogenic Gram-positive Bacillus sp. with top bud spore newly isolated from dying soft shell turtle. 16SrDNA sequencing showed that it is highly homologous to B. cereus, B. thuringiensis and B. anthracis. Biochemical examinations showed that it belongs to B. cereus. To further study the new pathogen, we conducted whole-genome sequencing based on single-molecular sequencing technology from PacBio. Genome assembly analysis showed that the strain has a 5,296,886 bp chromosome, a 218,649 bp plasmid and a 5221 bp plasmid with GC content of 35.51%, 31.91% and 29.75%, respectively. The genome contains 5736 coding sequences and 6 CRISPR systems located in the chromosome as well as 11 genomic islands in the chromosome and the large plasmid. Genome function analyses were annotated by nr database, SwissProt, KEGG, COG, GO, PHI, VFDB, ARDB, Secretory_Protein and T3SS. In addition, 13 gene clusters of secondary metabolism were predicted by antiSMASH. Comparison of SYJ15 with B. subtilis, B. anthracis, B. cereus and B. thuringiensis identified 1031 core genes of the five strains and 816 genes specific to SYJ15. In addition, SYJ15 had the most common core genes with B. thuringiensis, and the least with B. subtilis. Phylogenetic analysis demonstrated that SYJ15 is between B. thuringiensis and B. cereus, suggesting that SYJ15 belongs to Bacillus cereus group. We designed a specific primer pair to distinguish SYJ15 from B. pumilus, B. licheniformis, B.subtilis, B. thuringiensis and B. cereus. In conclusion, information of SYJ15 genome will help to enhance our understanding of pathogenesis of SYJ15 and find effective treatment.

Chromosome-scale genome assembly of kiwifruit Actinidia eriantha with single-molecule sequencing and chromatin interaction mapping.

BackgroundKiwifruit (Actinidia spp.) is a dioecious plant with fruits containing abundant vitamin C and minerals. A handful of kiwifruit species have been domesticated, among which Actinidiaeriantha is increasingly favored in breeding owing to its superior commercial traits. Recently, elite cultivars from A. eriantha have been successfully selected and further studies on their biology and breeding potential require genomic information, which is currently unavailable.FindingsWe assembled a chromosome-scale genome sequence of A. eriantha cultivar White using single-molecular sequencing and chromatin interaction map–based scaffolding. The assembly has a total size of 690.6 megabases and an N50 of 21.7 megabases. Approximately 99% of the assembly were in 29 pseudomolecules corresponding to the 29 kiwifruit chromosomes. Forty-three percent of the A. eriantha genome are repetitive sequences, and the non-repetitive part encodes 42,988 protein-coding genes, of which 39,075 have homologues from other plant species or protein domains. The divergence time between A. eriantha and its close relative Actinidia chinensis is estimated to be 3.3 million years, and after diversification, 1,727 and 1,506 gene families are expanded and contracted in A. eriantha, respectively.ConclusionsWe provide a high-quality reference genome for kiwifruit A. eriantha. This chromosome-scale genome assembly is substantially better than 2 published kiwifruit assemblies from A. chinensis in terms of genome contiguity and completeness. The availability of the A. eriantha genome provides a valuable resource for facilitating kiwifruit breeding and studies of kiwifruit biology.

Benchmark Evaluation of True Single Molecular Sequencing to Determine Cystic Fibrosis Airway Microbiome Diversity.

Cystic fibrosis (CF) is an autosomal recessive disease associated with recurrent lung infections that can lead to morbidity and mortality. The impact of antibiotics for treatment of acute pulmonary exacerbations on the CF airway microbiome remains unclear with prior studies giving conflicting results and being limited by their use of 16S ribosomal RNA sequencing. Our primary objective was to validate the use of true single molecular sequencing (tSMS) and PathoScope in the analysis of the CF airway microbiome. Three control samples were created with differing amounts of Burkholderia cepacia, Pseudomonas aeruginosa, and Prevotella melaninogenica, three common bacteria found in cystic fibrosis lungs. Paired sputa were also obtained from three study participants with CF before and >6 days after initiation of antibiotics. Antibiotic resistant B. cepacia and P. aeruginosa were identified in concurrently obtained respiratory cultures. Direct sequencing was performed using tSMS, and filtered reads were aligned to reference genomes from NCBI using PathoScope and Kraken and unique clade-specific marker genes using MetaPhlAn. A total of 180–518 K of 6–12 million filtered reads were aligned for each sample. Detection of known pathogens in control samples was most successful using PathoScope. In the CF sputa, alpha diversity measures varied based on the alignment method used, but similar trends were found between pre- and post-antibiotic samples. PathoScope outperformed Kraken and MetaPhlAn in our validation study of artificial bacterial community controls and also has advantages over Kraken and MetaPhlAn of being able to determine bacterial strains and the presence of fungal organisms. PathoScope can be confidently used when evaluating metagenomic data to determine CF airway microbiome diversity.

Progressive approach for SNP calling and haplotype assembly using single molecular sequencing data.

Haplotype information is essential to the complete description and interpretation of genomes, genetic diversity and genetic ancestry. The new technologies can provide Single Molecular Sequencing (SMS) data that cover about 90% of positions over chromosomes. However, the SMS data has a higher error rate comparing to 1% error rate for short reads. Thus, it becomes very difficult for SNP calling and haplotype assembly using SMS reads. Most existing technologies do not work properly for the SMS data. In this paper, we develop a progressive approach for SNP calling and haplotype assembly that works very well for the SMS data. Our method can handle more than 200 million non-N bases on Chromosome 1 with millions of reads, more than 100 blocks, each of which contains more than 2 million bases and more than 3K SNP sites on average. Experiment results show that the false discovery rate and false negative rate for our method are 15.7 and 11.0% on NA12878, and 16.5 and 11.0% on NA24385. Moreover, the overall switch errors for our method are 7.26 and 5.21 with average 3378 and 5736 SNP sites per block on NA12878 and NA24385, respectively. Here, we demonstrate that SMS reads alone can generate a high quality solution for both SNP calling and haplotype assembly. Source codes and results are available at https://github.com/guofeieileen/SMRT/wiki/Software.

Filling reference gaps via assembling DNA barcodes using high-throughput sequencing-moving toward barcoding the world.

Over the past decade, biodiversity researchers have dedicated tremendous efforts to constructing DNA reference barcodes for rapid species registration and identification. Although analytical cost for standard DNA barcoding has been significantly reduced since early 2000, further dramatic reduction in barcoding costs is unlikely because Sanger sequencing is approaching its limits in throughput and chemistry cost. Constraints in barcoding cost not only led to unbalanced barcoding efforts around the globe, but also prevented high-throughput sequencing (HTS)–based taxonomic identification from applying binomial species names, which provide crucial linkages to biological knowledge. We developed an Illumina-based pipeline, HIFI-Barcode, to produce full-length Cytochrome c oxidase subunit I (COI) barcodes from pooled polymerase chain reaction amplicons generated by individual specimens. The new pipeline generated accurate barcode sequences that were comparable to Sanger standards, even for different haplotypes of the same species that were only a few nucleotides different from each other. Additionally, the new pipeline was much more sensitive in recovering amplicons at low quantity. The HIFI-Barcode pipeline successfully recovered barcodes from more than 78% of the polymerase chain reactions that didn’t show clear bands on the electrophoresis gel. Moreover, sequencing results based on the single molecular sequencing platform Pacbio confirmed the accuracy of the HIFI-Barcode results. Altogether, the new pipeline can provide an improved solution to produce full-length reference barcodes at about one-tenth of the current cost, enabling construction of comprehensive barcode libraries for local fauna, leading to a feasible direction for DNA barcoding global biomes.

Identifying and sequencing a Mycobacterium sp. strain F4 as a potential bioremediation agent for quinclorac.

Quinclorac is a widely used herbicide in rice filed. Unfortunately, quinclorac residues are phytotoxic to many crops/vegetables. The degradation of quinclorac in nature is very slow. On the other hand, degradation of quinclorac using bacteria can be an effective and efficient method to reduce its contamination. In this study, we isolated a quinclorac bioremediation bacterium strain F4 from quinclorac contaminated soils. Based on morphological characteristics and 16S rRNA gene sequence analysis, we identified strain F4 as Mycobacterium sp. We investigated the effects of temperature, pH, inoculation size and initial quinclorac concentration on growth and degrading efficiency of F4 and determined the optimal quinclorac degrading condition of F4. Under optimal degrading conditions, F4 degraded 97.38% of quinclorac from an initial concentration of 50 mg/L in seven days. Our indoor pot experiment demonstrated that the degradation products were non-phytotoxic to tobacco. After analyzing the quinclorac degradation products of F4, we proposed that F4 could employ two pathways to degrade quinclorac: one is through methylation, the other is through dechlorination. Furthermore, we reconstructed the whole genome of F4 through single molecular sequencing and de novo assembly. We identified 77 methyltransferases and eight dehalogenases in the F4 genome to support our hypothesized degradation path.

Hybrid de novo genome assembly of the Chinese herbal plant danshen (Salvia miltiorrhiza Bunge).

BackgroundDanshen (Salvia miltiorrhiza Bunge), also known as Chinese red sage, is a member of Lamiaceae family. It is valued in traditional Chinese medicine, primarily for the treatment of cardiovascular and cerebrovascular diseases. Because of its pharmacological potential, ongoing research aims to identify novel bioactive compounds in danshen, and their biosynthetic pathways. To date, only expressed sequence tag (EST) and RNA-seq data for this herbal plant are available to the public. We therefore propose that the construction of a reference genome for danshen will help elucidate the biosynthetic pathways of important secondary metabolites, thereby advancing the investigation of novel drugs from this plant.FindingsWe assembled the highly heterozygous danshen genome with the help of 395 × raw read coverage using Illumina technologies and about 10 × raw read coverage by using single molecular sequencing technology. The final draft genome is approximately 641 Mb, with a contig N50 size of 82.8 kb and a scaffold N50 size of 1.2 Mb. Further analyses predicted 34,598 protein-coding genes and 1,644 unique gene families in the danshen genome.ConclusionsThe draft danshen genome will provide a valuable resource for the investigation of novel bioactive compounds in this Chinese herb.Electronic supplementary materialThe online version of this article (doi:10.1186/s13742-015-0104-3) contains supplementary material, which is available to authorized users.

Current status on single molecular sequencing based on protein nanopores

In the 1960s, Hladky and Haydon have succeeded in detecting ionic flows across thin lipid membrane when adding small lipid-soluble molecules such as surface-active polypeptides to artificial membrane, which has laid the foundation for decoding single strand DNA when it crosses the nanopore under an applied electric field. In this paper, we will review several kinds of protein nanopores used in single molecular sequencing, such as α-hemolysin nanopore, MspA nanopore and membrane-adapted phi29 motor protein nanopore.

Advances in DNA sequencing: Challenges and limitations of personal sequencing

The advent of second and third generation DNA sequencing techniqnologies have revolutionized the genomics research. The Single Molecule Sequencing technologies are adequate to minimize occurrence of errors in sequencing. The third generation sequencing technologies could overcome limitations of first and second-generation sequencing technologies. The almost complete human genome sequencing was performed by using single molecular sequencing technologies. Benefits, risks and safeguards associated with the disclosure of information of very intimate kind contained in human genome, were also discussed. The power of whole sequencing for human welfare arises only from associations with medical histories, behavioral characteristics, physical descriptions and genome-environment interactions not from mere knowledge of base pairs. Key words: DNA sequencing, medical histories, behavioral characteristics, physical descriptions.