Pacific Biosciences Sequencing Platform Research Articles

HLA/MHC and KIR characterization in humans and non-human primates using Oxford Nanopore Technologies and Pacific Biosciences sequencing platforms.

The gene products of the HLA/MHC and KIR multigene families are important modulators of the immune system and are associated with health and disease. Characterization of the genes encoding these receptors has been integrated into different biomedical applications, including transplantation and reproduction biology, immune therapies and in fundamental research into disease susceptibility or resistance. Conventional short-read sequencing strategies have shown their value in high throughput typing, but are insufficient to uncover the entire complexity of the highly polymorphic HLA/MHC and KIR gene systems. The implementation of single-molecule and real-time sequencing platforms, offered by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), revolutionized the fields of genomics and transcriptomics. Using fundamentally distinct principles, these platforms generate long-read data that can unwire the plasticity of the HLA/MHC and KIR genes, including high-resolution characterization of genes, alleles, phased haplotypes, transcription levels and epigenetics modification patterns. These insights might have profound clinical relevance, such as improved matching of donors and patients in clinical transplantation, but could also lift disease association studies to a higher level. Even more, a comprehensive characterization may refine animal models in preclinical studies. In this review, the different HLA/MHC and KIR characterization approaches using PacBio and ONT platforms are described and discussed.

Comparison of the Oral Microbiota Structure among People from the Same Ethnic Group Living in Different Environments.

The characteristics of the oral microbiota may depend on oral health, age, diet, and geography, but the influence of the geographic setting on the oral microbiota has received limited attention. The characteristics of oral microbiota have been reported to differ between urban and rural environments. In order to minimize the influence of genetic background, we recruited 54 volunteers from the same ethnic group, living in urban and rural areas of Gansu Province, China. We collected dental plaque samples and divided them into four groups according to the participant's area of residence and dental caries status. We sequenced the 16S rRNA of these samples using the Pacific Biosciences sequencing platform and analyzed the correlation between the geographic area and the characteristics of the oral microbiota. Analysis of the alpha and beta diversity revealed that there were significant differences in diversity and composition of dental plaque microflora among the four groups. Cluster analysis revealed that geographic area played an important role in determining the oral microbiota. Network analysis of oral microorganisms showed that geographic differences had major influence on the composition characteristics and internal structure of oral microorganisms. We found that some dominant strains which may play a key role in maintaining oral health, such as Streptococcus oralis, Capnocytophaga sputigena, Porphyromonas catoniae, Corynebacterium matruchotii, Haemophilus parainfluenzae, and Prevotella loescheii, were less affected by the geographic setting. These results provide a deeper understanding of factors influencing the composition of the oral microbiota and could contribute to early diagnosis and effective prevention of dental caries in different settings.

Production of polyhydroxyalkanoates from propylene oxide saponification wastewater residual sludge using volatile fatty acids and bacterial community succession

The active sludge treating propylene oxide saponification wastewater has heavy salt concentration and is hard to treat. The integration of the residual sludge treatment with polyhydroxyalkanoates (PHA) production may provide an economic and environment friendly solution. PHA production was therefore studied in two sequencing biological reactors with effective volume of 30 L using the active sludge. The two reactors, named as SBR-I and SBR-II, were fed with acetic acid, and a mixture of acetic acid and propionic acid respectively. PHA was obtained with a yield of 9.257 g/L in SBR-II. Also, the proportion of 3-hydroxyvalarate was enhanced from 5% to 30% in comparison to SBR-I (5.471 g/L). Illumina MiSeq and Pacific Biosciences sequencing platforms were used to evaluate the community structure, which revealed that the bacterial genera showed a high degree of diversity in the PHA accumulating microbial community. Azoarcus was the most dominant PHA accumulating microorganism after acclimation.

Genome sequencing, assembly, and annotation of the self-flocculating microalga Scenedesmus obliquus AS-6-11

BackgroundScenedesmus obliquus belongs to green microalgae and is widely used in aquaculture as feed, which is also explored for lipid production and bioremediation. However, genomic studies of this microalga have been very limited. Cell self-flocculation of microalgal cells can be used as a simple and economic method for harvesting biomass, and it is of great importance to perform genome-scale studies for the self-flocculating S. obliquus strains to promote their biotechnological applications.ResultsWe employed the Pacific Biosciences sequencing platform for sequencing the genome of the self-flocculating microalga S. obliquus AS-6-11, and used the MECAT software for de novo genome assembly. The estimated genome size of S. obliquus AS-6-11 is 172.3 Mbp with an N50 of 94,410 bp, and 31,964 protein-coding genes were identified. Gene Ontology (GO) and KEGG pathway analyses revealed 65 GO terms and 428 biosynthetic pathways. Comparing to the genome sequences of the well-studied green microalgae Chlamydomonas reinhardtii, Chlorella variabilis, Volvox carteri and Micractinium conductrix, the genome of S. obliquus AS-6-11 encodes more unique proteins, including one gene that encodes D-mannose binding lectin. Genes encoding the glycosylphosphatidylinositol (GPI)-anchored cell wall proteins, and proteins with fasciclin domains that are commonly found in cell wall proteins might be responsible for the self-flocculating phenotype, and were analyzed in detail. Four genes encoding both GPI-anchored cell wall proteins and fasciclin domain proteins are the most interesting targets for further studies.ConclusionsThe genome sequence of the self-flocculating microalgal S. obliquus AS-6-11 was annotated and analyzed. To our best knowledge, this is the first report on the in-depth annotation of the S. obliquus genome, and the results will facilitate functional genomic studies and metabolic engineering of this important microalga. The comparative genomic analysis here also provides new insights into the evolution of green microalgae. Furthermore, identification of the potential genes encoding self-flocculating proteins will benefit studies on the molecular mechanism underlying this phenotype for its better control and biotechnological applications as well.

A Refined View of Airway Microbiome in Chronic Obstructive Pulmonary Disease at Species and Strain-Levels.

Little is known about the underlying airway microbiome diversity in chronic obstructive pulmonary disease (COPD) at in-depth taxonomic levels. Here we present the first insights on the COPD airway microbiome at species and strain-levels. The full-length 16S rRNA gene was characterized from sputum in 98 COPD patients and 27 age-matched healthy controls, using the Pacific Biosciences sequencing platform. Individual species within the same genus exhibited reciprocal relationships with COPD and disease severity. Species dominant in health can be taken over by another species within the same genus but with potentially increasing pathogenicity in severe COPD patients. Ralstonia mannitolilytica, an opportunistic pathogen, was significantly increased in frequent exacerbators (fold-change = 4.94, FDR P = 0.005). There were distinct patterns of interaction between bacterial species and host inflammatory mediators according to neutrophilic or eosinophilic inflammations, two major airway inflammatory phenotypes in COPD. Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, and Neisseria meningitidis were associated with enhanced Th1, Th17 and pro-inflammatory mediators, while a group of seven species including Tropheryma whipplei were specifically associated with Th2 mediators related to eosinophilia. We developed an automated pipeline to assign strain-level taxonomy leveraging bacterial intra-genomic 16S allele frequency. Using this pipeline we further resolved three non-typeable H. influenzae strains PittEE, PittGG and 86-028NP with reasonable precision and uncovered strain-level variation related to airway inflammation. In particular, 86-028NP and PittGG strains exhibited inverse associations with Th2 chemokines CCL17 and CCL13, suggesting their abundances may inversely predict eosinophilic inflammation. A systematic comparison of 16S hypervariable regions indicated V1V3 instead of the commonly used V4 region was the best surrogate for airway microbiome. The full-length 16S data augmented the power of functional inference, which slightly better recapitulated the actual metagenomes. This led to the unique identification of butyrate-producing and nitrate reduction pathways as depleted in COPD. Our analysis uncovered finer-scale airway microbial diversity that was previously underappreciated, thus enabled a refined view of the airway microbiome in COPD.

A chromosomal-level genome assembly for the giant African snail Achatina fulica.

Background Achatina fulica, the giant African snail, is the largest terrestrial mollusk species. Owing to its voracious appetite, wide environmental adaptability, high growth rate, and reproductive capacity, it has become an invasive species across the world, mainly in Southeast Asia, Japan, the western Pacific islands, and China. This pest can damage agricultural crops and is an intermediate host of many parasites that can threaten human health. However, genomic information of A. fulica remains limited, hindering genetic and genomic studies for invasion control and management of the species.FindingsUsing a k-mer–based method, we estimated the A. fulica genome size to be 2.12 Gb, with a high repeat content up to 71%. Roughly 101.6 Gb genomic long-read data of A. fulica were generated from the Pacific Biosciences sequencing platform and assembled to produce a first A. fulica genome of 1.85 Gb with a contig N50 length of 726 kb. Using contact information from the Hi-C sequencing data, we successfully anchored 99.32% contig sequences into 31 chromosomes, leading to the final contig and scaffold N50 length of 721 kb and 59.6 Mb, respectively. The continuity, completeness, and accuracy were evaluated by genome comparison with other mollusk genomes, BUSCO assessment, and genomic read mapping. A total of 23,726 protein-coding genes were predicted from the assembled genome, among which 96.34% of the genes were functionally annotated. The phylogenetic analysis using whole-genome protein-coding genes revealed that A. fulica separated from a common ancestor with Biomphalaria glabrata ∼182 million years ago.ConclusionTo our knowledge, the A. fulica genome is the first terrestrial mollusk genome published to date. The chromosome sequence of A. fulica will provide the research community with a valuable resource for population genetics and environmental adaptation studies for the species, as well as investigations of the chromosome-level of evolution within mollusks.

A draft genome assembly of halophyte Suaeda aralocaspica, a plant that performs C4 photosynthesis within individual cells.

BackgroundThe halophyte Suaeda aralocaspica performs complete C4 photosynthesis within individual cells (SCC4), which is distinct from typical C4 plants, which require the collaboration of 2 types of photosynthetic cells. However, despite SCC4 plants having features that are valuable in engineering higher photosynthetic efficiencies in agriculturally important C3 species such as rice, there are no reported sequenced SCC4 plant genomes, limiting our understanding of the mechanisms involved in, and evolution of, SCC4 photosynthesis.FindingsUsing Illumina and Pacific Biosciences sequencing platforms, we generated ∼202 Gb of clean genomic DNA sequences having a 433-fold coverage based on the 467 Mb estimated genome size of S. aralocaspica. The final genome assembly was 452 Mb, consisting of 4,033 scaffolds, with a scaffold N50 length of 1.83 Mb. We annotated 29,604 protein-coding genes using Evidence Modeler based on the gene information from ab initio predictions, homology levels with known genes, and RNA sequencing–based transcriptome evidence. We also annotated noncoding genes, including 1,651 long noncoding RNAs, 21 microRNAs, 382 transfer RNAs, 88 small nuclear RNAs, and 325 ribosomal RNAs. A complete (circular with no gaps) chloroplast genome of S. aralocaspica 146,654 bp in length was also assembled.ConclusionsWe have presented the genome sequence of the SCC4 plant S. aralocaspica. Knowledge of the genome of S. aralocaspica should increase our understanding of the evolution of SCC4 photosynthesis and contribute to the engineering of C4 photosynthesis into economically important C3 crops.

NanoAmpli-Seq: a workflow for amplicon sequencing for mixed microbial communities on the nanopore sequencing platform.

BackgroundAmplicon sequencing on Illumina sequencing platforms leverages their deep sequencing and multiplexing capacity but is limited in genetic resolution due to short read lengths. While Oxford Nanopore or Pacific Biosciences sequencing platforms overcome this limitation, their application has been limited due to higher error rates or lower data output.ResultsIn this study, we introduce an amplicon sequencing workflow, i.e., NanoAmpli-Seq, that builds on the intramolecular-ligated nanopore consensus sequencing (INC-Seq) approach and demonstrate its application for full-length 16S rRNA gene sequencing. NanoAmpli-Seq includes vital improvements to the INC-Seq protocol that reduces sample processing time while significantly improving sequence accuracy. The developed protocol includes chopSeq software for fragmentation and read orientation correction of INC-Seq consensus reads while nanoClust algorithm was designed for read partitioning-based de novo clustering and within cluster consensus calling to obtain accurate full-length 16S rRNA gene sequences.ConclusionsNanoAmpli-Seq accurately estimates the diversity of tested mock communities with average consensus sequence accuracy of 99.5% for 2D and 1D2 sequencing on the nanopore sequencing platform. Nearly all residual errors in NanoAmpli-Seq sequences originate from deletions in homopolymer regions, indicating that homopolymer aware base calling or error correction may allow for sequencing accuracy comparable to short-read sequencing platforms.

Chromosome-level reference genome and alternative splicing atlas of moso bamboo (Phyllostachys edulis)

BackgroundBamboo is one of the most important nontimber forestry products worldwide. However, a chromosome-level reference genome is lacking, and an evolutionary view of alternative splicing (AS) in bamboo remains unclear despite emerging omics data and improved technologies.ResultsHere, we provide a chromosome-level de novo genome assembly of moso bamboo (Phyllostachys edulis) using additional abundance sequencing data and a Hi-C scaffolding strategy. The significantly improved genome is a scaffold N50 of 79.90 Mb, approximately 243 times longer than the previous version. A total of 51,074 high-quality protein-coding loci with intact structures were identified using single-molecule real-time sequencing and manual verification. Moreover, we provide a comprehensive AS profile based on the identification of 266,711 unique AS events in 25,225 AS genes by large-scale transcriptomic sequencing of 26 representative bamboo tissues using both the Illumina and Pacific Biosciences sequencing platforms. Through comparisons with orthologous genes in related plant species, we observed that the AS genes are concentrated among more conserved genes that tend to accumulate higher transcript levels and share less tissue specificity. Furthermore, gene family expansion, abundant AS, and positive selection were identified in crucial genes involved in the lignin biosynthetic pathway of moso bamboo.ConclusionsThese fundamental studies provide useful information for future in-depth analyses of comparative genome and AS features. Additionally, our results highlight a global perspective of AS during evolution and diversification in bamboo.

SMRT Gate: A method for validation of synthetic constructs on Pacific Biosciences sequencing platforms.

Current DNA assembly methods are prone to sequence errors, requiring rigorous quality control (QC) to identify incorrect assemblies or synthesized constructs. Such errors can lead to misinterpretation of phenotypes. Because of this intrinsic problem, routine QC analysis is generally performed on three or more clones using a combination of restriction endonuclease assays, colony PCR, and Sanger sequencing. However, as new automation methods emerge that enable high-throughput assembly, QC using these techniques has become a major bottleneck. Here, we describe a quick and affordable methodology for the QC of synthetic constructs. Our method involves a one-pot digestion-ligation DNA assembly reaction, based on the Golden Gate assembly methodology, that is coupled with Pacific Biosciences' Single Molecule, Real-Time (PacBio SMRT) sequencing technology.

Diversity of the TLR4 Immunity Receptor in Czech Native Cattle Breeds Revealed Using the Pacific Biosciences Sequencing Platform

ABSTRACTThe allelic variants of immunity genes in historical breeds likely reflect local infection pressure and therefore represent a reservoir for breeding. Screening to determine the diversity of the Toll-like receptor gene TLR4 was conducted in two conserved cattle breeds: Czech Red and Czech Red Pied. High-throughput sequencing of pooled PCR amplicons using the PacBio platform revealed polymorphisms, which were subsequently confirmed via genotyping techniques. Eight SNPs found in coding and adjacent regions were grouped into 18 haplotypes, representing a significant portion of the known diversity in the global breed panel and presumably exceeding diversity in production populations. Notably, the ancient Czech Red breed appeared to possess greater haplotype diversity than the Czech Red Pied breed, a Simmental variant, although the haplotype frequencies might have been distorted by significant crossbreeding and bottlenecks in the history of Czech Red cattle. The differences in haplotype frequencies validated the phenotypic distinctness of the local breeds. Due to the availability of Czech Red Pied production herds, the effect of intensive breeding on TLR diversity can be evaluated in this model. The advantages of the Pacific Biosciences technology for the resequencing of long PCR fragments with subsequent direct phasing were independently validated.

Metataxonomics reveal vultures as a reservoir for Clostridium perfringens

The Old World vulture may carry and spread pathogens for emerging infections since they feed on the carcasses of dead animals and participate in the sky burials of humans, some of whom have died from communicable diseases. Therefore, we studied the precise fecal microbiome of the Old World vulture with metataxonomics, integrating the high-throughput sequencing of almost full-length small subunit ribosomal RNA (16S rRNA) gene amplicons in tandem with the operational phylogenetic unit (OPU) analysis strategy. Nine vultures of three species were sampled using rectal swabs on the Qinghai-Tibet Plateau, China. Using the Pacific Biosciences sequencing platform, we obtained 54 135 high-quality reads of 16S rRNA amplicons with an average of 1442±6.9 bp in length and 6015±1058 reads per vulture. Those sequences were classified into 314 OPUs, including 102 known species, 50 yet to be described species and 161 unknown new lineages of uncultured representatives. Forty-five species have been reported to be responsible for human outbreaks or infections, and 23 yet to be described species belong to genera that include pathogenic species. Only six species were common to all vultures. Clostridium perfringens was the most abundant and present in all vultures, accounting for 30.8% of the total reads. Therefore, using the new technology, we found that vultures are an important reservoir for C. perfringens as evidenced by the isolation of 107 strains encoding for virulence genes, representing 45 sequence types. Our study suggests that the soil-related C. perfringens and other pathogens could have a reservoir in vultures and other animals.Emerging Microbes & Infections (2017) 6, e9; doi:10.1038/emi.2016.137; published online 22 February 2017

Microbial phylogenetic profiling with the Pacific Biosciences sequencing platform

High-throughput sequencing of 16S rRNA gene amplicons has revolutionized the capacity and depth of microbial community profiling. Several sequencing platforms are available, but most phylogenetic studies are performed on the 454-pyrosequencing platform because its longer reads can give finer phylogenetic resolution. The Pacific Biosciences (PacBio) sequencing platform is significantly less expensive per run, does not rely on amplification for library generation, and generates reads that are, on average, four times longer than those from 454 (C2 chemistry), but the resulting high error rates appear to preclude its use in phylogenetic profiling. Recently, however, the PacBio platform was used to characterize four electrosynthetic microbiomes to the genus-level for less than USD 1,000 through the use of PacBio’s circular consensus sequence technology. Here, we describe in greater detail: 1) the output from successful 16S rRNA gene amplicon profiling with PacBio, 2) how the analysis was contingent upon several alterations to standard bioinformatic quality control workflows, and 3) the advantages and disadvantages of using the PacBio platform for community profiling.