Full-length Reference Sequences Research Articles

Identification and Molecular Characterization of Four New Blastocystis Subtypes Designated ST35-ST38.

Three recent studies of Blastocystis epidemiology in mammalian hosts identified four novel sequences that appeared to share B. lapemi as the most similar sequence. However, full-length ssu rRNA gene sequences were not available to confirm the validity of these new subtypes. In the present study, Nanopore MinION sequencing was used to obtain full-length reference sequences for each of the new subtypes. Additionally, phylogenetic analyses and pairwise distance comparisons were performed to confirm the validity of each of these new subtypes. We propose that the novel sequences described in this study should be assigned the subtype designations ST35-ST38. The full-length reference sequences of ST35-ST38 will assist in accurate sequence descriptions in future studies of Blastocystis epidemiology and subtype diversity.

Mind the Gap: New Full-Length Sequences of Blastocystis Subtypes Generated via Oxford Nanopore Minion Sequencing Allow for Comparisons between Full-Length and Partial Sequences of the Small Subunit of the Ribosomal RNA Gene.

Blastocystis is a common food- and water-borne intestinal protist parasite of humans and many other animals. Blastocystis comprises multiple subtypes (STs) based on variability within the small subunit ribosomal (SSU rRNA) RNA gene. Though full-length reference sequences of the SSU rRNA gene are a current requirement to name a novel Blastocystis subtype, full-length reference sequences are not currently available for all subtypes. In the present study, Oxford Nanopore MinION long-read sequencing was employed to generate full-length SSU rRNA sequences for seven new Blastocystis subtypes for which no full-length references currently exist: ST21, ST23, ST24, ST25, ST26, ST27, and ST28. Phylogenetic analyses and pairwise distance matrixes were used to compare full-length and partial sequences of the two regions that are most commonly used for subtyping. Analyses included Blastocystis nucleotide sequences obtained in this study (ST21 and ST23–ST28) and existing subtypes for which full-length reference sequences were available (ST1–ST17 and ST29). The relationships and sequence variance between new and existing subtypes observed in analyses of different portions of the SSU rRNA gene are discussed. The full-length SSU rRNA reference sequences generated in this study provide essential new data to study and understand the relationships between the genetic complexity of Blastocystis and its host specificity, pathogenicity, and epidemiology.

Use of Oxford Nanopore MinION to generate full-length sequences of the Blastocystis small subunit (SSU) rRNA gene

BackgroundBlastocystis sp. is one of the most common enteric parasites of humans and animals worldwide. It is well recognized that this ubiquitous protist displays a remarkable degree of genetic diversity in the SSU rRNA gene, which is currently the main gene used for defining Blastocystis subtypes. Yet, full-length reference sequences of this gene are available for only 16 subtypes of Blastocystis in part because of the technical difficulties associated with obtaining these sequences from complex samples.MethodsWe have developed a method using Oxford Nanopore MinION long-read sequencing and universal eukaryotic primers to produce full-length (> 1800 bp) SSU rRNA gene sequences for Blastocystis. Seven Blastocystis specimens representing five subtypes (ST1, ST4, ST10, ST11, and ST14) obtained both from cultures and feces were used for validation.ResultsWe demonstrate that this method can be used to produce highly accurate full-length sequences from both cultured and fecal DNA isolates. Full-length sequences were successfully obtained from all five subtypes including ST11 for which no full-length reference sequence currently exists and for an isolate that contained mixed ST10/ST14.ConclusionsThe suitability of the use of MinION long-read sequencing technology to successfully generate full-length Blastocystis SSU rRNA gene sequences was demonstrated. The ability to produce full-length SSU rRNA gene sequences is key in understanding the role of genetic diversity in important aspects of Blastocystis biology such as transmission, host specificity, and pathogenicity.Graphical

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.

P103 The promise of cost-efficient full-length HLA class I genotyping: Advances using nanopore sequencing

Aim Nanopore based sequencing has seen rapid advancement in the recent years with iterations in pore structure, sequencing chemistries and base callers. This has led to increasingly accurate sequencing of extremely long DNA molecules. Since current HLA genotyping algorithms are not optimized for nanopore data we developed a genotyping algorithm based on read grouping and subsequent mapping to a reduced reference database. Methods High raw sequencing errors present a challenge for genotyping based on direct mapping to a reference database. Our algorithm, Poretyper, obviates this initial mapping by first grouping the raw reads based on the distributions of k-mers within each read. A multiple sequence alignment derived from the groups of raw reads results in a set of consensus sequences which represent potential alleles. These consensus sequences are then used to create a culled reference database dramatically reducing the search space, thus reducing artefactual raw read mappings. Results HLA-A, HLA-B and HLA-C for a hundred samples drawn from the DKMS donor registry were sequenced using the MinION with R9.5 chemistry. All hundred samples were then genotyped using Poretyper and existing G-group pretypings could be recapitulated failing only for those alleles where no full length sequences were available. Conclusions Nanopore sequencing presents a viable and accurate platform for cost-efficient full-length HLA Class I genotyping. For those alleles where full length reference sequences are not available, an in silico extension of such allele sequences using the full-length sequence of the next closest allele presents a viable approach for full-length genotyping.

P062Coding and non-coding variation in 60 novel full-length sequences of KIR2DL1

Aim KIR2DL1 is a member of the killer-cell immunoglobulin-like receptor (KIR) family, which is an important factor of the human immune system. KIR genes are key regulators of natural killer cell activity and partly bind to proteins of the human leukocyte antigen (HLA) family, e.g. HLA-B and -C in the case of KIR2DL1. Likely due to the complexity of the KIR locus with its extensive genetic variation, only little is known about the impact of allelic variation. Here, we identified novel KIR2DL1 alleles by routine high-throughput exon-based KIR genotyping and subsequently created full-length reference sequences. We analysed coding and non-coding variation to identify possible systematic differences between alleles. Methods We sequenced whole 16 kb amplicons of KIR2DL1 using shotgun sequencing (Illumina MiSeq) and single molecule real time (SMRT) sequencing (PacBio Sequel). Using the R package DR2S, we combined phase information from SMRT sequencing with the accuracy of shotgun sequencing to generate phased full-length sequences. Results We successfully generated 60 distinct KIR2DL1 allele sequences from 45 specifically selected samples. This includes 41 novel alleles and 17 distinct alleles that were previously only partially characterised. The sequence analysis revealed three deep allelic groups separated by 164 variable positions. Allelic variants of two groups harbour mutually exclusive nucleotides, while a third group of alleles may harbour nucleotides of both the other groups. The three groups are mostly separated by non-coding SNPs, indels, and a T homopolymer of fixed length in one and variable length in two groups. Some separating variants exist in most exons and alter the amino acid sequence of the leader peptide, the extracellular D1- and D2-domains containing the HLA binding sites as well as the cytoplasmatic domain. Conclusions Our sequencing efforts resulted in a 4-fold increase in known full-length sequences of KIR2DL1, enabling further research on this KIR gene. We gained insights into systematic differences at the sequence level which might be responsible for or indicative of medically relevant allelic differences. Especially variation in the D2-domain has been shown to be involved in binding to HLA proteins and may as such be clinically relevant.

Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system.

Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina’s MiSeq, have allowed researchers to obtain millions of high quality but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3–V5, V1–V3, V1–V5, V1–V6, and V1–V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1–V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina’s MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting.

The feline oral microbiome: A provisional 16S rRNA gene based taxonomy with full-length reference sequences

The human oral microbiome is known to play a significant role in human health and disease. While less well studied, the feline oral microbiome is thought to play a similarly important role. To determine roles oral bacteria play in health and disease, one first has to be able to accurately identify bacterial species present. 16S rRNA gene sequence information is widely used for molecular identification of bacteria and is also useful for establishing the taxonomy of novel species.The objective of this research was to obtain full 16S rRNA gene reference sequences for feline oral bacteria, place the sequences in species-level phylotypes, and create a curated 16S rRNA based taxonomy for common feline oral bacteria.Clone libraries were produced using “universal” and phylum-selective PCR primers and DNA from pooled subgingival plaque from healthy and periodontally diseased cats. Bacteria in subgingival samples were also cultivated to obtain isolates. Full-length 16S rDNA sequences were determined for clones and isolates that represent 171 feline oral taxa. A provisional curated taxonomy was developed based on the position of each taxon in 16S rRNA phylogenetic trees.The feline oral microbiome curated taxonomy and 16S rRNA gene reference set will allow investigators to refer to precisely defined bacterial taxa. A provisional name such as “Propionibacterium sp. feline oral taxon FOT-327” is an anchor to which clone, strain or GenBank names or accession numbers can point. Future next-generation-sequencing studies of feline oral bacteria will be able to map reads to taxonomically curated full-length 16S rRNA gene sequences.

LTRsift: a graphical user interface for semi-automatic classification and postprocessing of de novo detected LTR retrotransposons

BackgroundLong terminal repeat (LTR) retrotransposons are a class of eukaryotic mobile elements characterized by a distinctive sequence similarity-based structure. Hence they are well suited for computational identification. Current software allows for a comprehensive genome-wide de novo detection of such elements. The obvious next step is the classification of newly detected candidates resulting in (super-)families. Such a de novo classification approach based on sequence-based clustering of transposon features has been proposed before, resulting in a preliminary assignment of candidates to families as a basis for subsequent manual refinement. However, such a classification workflow is typically split across a heterogeneous set of glue scripts and generic software (for example, spreadsheets), making it tedious for a human expert to inspect, curate and export the putative families produced by the workflow.ResultsWe have developed LTRsift, an interactive graphical software tool for semi-automatic postprocessing of de novo predicted LTR retrotransposon annotations. Its user-friendly interface offers customizable filtering and classification functionality, displaying the putative candidate groups, their members and their internal structure in a hierarchical fashion. To ease manual work, it also supports graphical user interface-driven reassignment, splitting and further annotation of candidates. Export of grouped candidate sets in standard formats is possible. In two case studies, we demonstrate how LTRsift can be employed in the context of a genome-wide LTR retrotransposon survey effort.ConclusionsLTRsift is a useful and convenient tool for semi-automated classification of newly detected LTR retrotransposons based on their internal features. Its efficient implementation allows for convenient and seamless filtering and classification in an integrated environment. Developed for life scientists, it is helpful in postprocessing and refining the output of software for predicting LTR retrotransposons up to the stage of preparing full-length reference sequence libraries. The LTRsift software is freely available at http://www.zbh.uni-hamburg.de/LTRsift under an open-source license.

Profiling the venom gland transcriptomes of Costa Rican snakes by 454 pyrosequencing

BackgroundA long term research goal of venomics, of applied importance for improving current antivenom therapy, but also for drug discovery, is to understand the pharmacological potential of venoms. Individually or combined, proteomic and transcriptomic studies have demonstrated their feasibility to explore in depth the molecular diversity of venoms. In the absence of genome sequence, transcriptomes represent also valuable searchable databases for proteomic projects.ResultsThe venom gland transcriptomes of 8 Costa Rican taxa from 5 genera (Crotalus, Bothrops, Atropoides, Cerrophidion, and Bothriechis) of pitvipers were investigated using high-throughput 454 pyrosequencing. 100,394 out of 330,010 masked reads produced significant hits in the available databases. 5.165,220 nucleotides (8.27%) were masked by RepeatMasker, the vast majority of which corresponding to class I (retroelements) and class II (DNA transposons) mobile elements. BLAST hits included 79,991 matches to entries of the taxonomic suborder Serpentes, of which 62,433 displayed similarity to documented venom proteins. Strong discrepancies between the transcriptome-computed and the proteome-gathered toxin compositions were obvious at first sight. Although the reasons underlaying this discrepancy are elusive, since no clear trend within or between species is apparent, the data indicate that individual mRNA species may be translationally controlled in a species-dependent manner. The minimum number of genes from each toxin family transcribed into the venom gland transcriptome of each species was calculated from multiple alignments of reads matched to a full-length reference sequence of each toxin family. Reads encoding ORF regions of Kazal-type inhibitor-like proteins were uniquely found in Bothriechis schlegelii and B. lateralis transcriptomes, suggesting a genus-specific recruitment event during the early-Middle Miocene. A transcriptome-based cladogram supports the large divergence between A. mexicanus and A. picadoi, and a closer kinship between A. mexicanus and C. godmani.ConclusionsOur comparative next-generation sequencing (NGS) analysis reveals taxon-specific trends governing the formulation of the venom arsenal. Knowledge of the venom proteome provides hints on the translation efficiency of toxin-coding transcripts, contributing thereby to a more accurate interpretation of the transcriptome. The application of NGS to the analysis of snake venom transcriptomes, may represent the tool for opening the door to systems venomics.

Mapping the protistan 'rare biosphere'

The use of cultivation-independent approaches to map microbial diversity, including recent work published in BMC Biology, has now shown that protists, like bacteria/archaea, are much more diverse than had been realized. Uncovering eukaryotic diversity may now be limited not by access to samples or cost but rather by the availability of full-length reference sequence data.See research article http://www.biomedcentral.com/1741-7007/7/72

Comparison of methods for genomic localization of gene trap sequences

BackgroundGene knockouts in a model organism such as mouse provide a valuable resource for the study of basic biology and human disease. Determining which gene has been inactivated by an untargeted gene trapping event poses a challenging annotation problem because gene trap sequence tags, which represent sequence near the vector insertion site of a trapped gene, are typically short and often contain unresolved residues. To understand better the localization of these sequences on the mouse genome, we compared stand-alone versions of the alignment programs BLAT, SSAHA, and MegaBLAST. A set of 3,369 sequence tags was aligned to build 34 of the mouse genome using default parameters for each algorithm. Known genome coordinates for the cognate set of full-length genes (1,659 sequences) were used to evaluate localization results.ResultsIn general, all three programs performed well in terms of localizing sequences to a general region of the genome, with only relatively subtle errors identified for a small proportion of the sequence tags. However, large differences in performance were noted with regard to correctly identifying exon boundaries. BLAT correctly identified the vast majority of exon boundaries, while SSAHA and MegaBLAST missed the majority of exon boundaries. SSAHA consistently reported the fewest false positives and is the fastest algorithm. MegaBLAST was comparable to BLAT in speed, but was the most susceptible to localizing sequence tags incorrectly to pseudogenes.ConclusionThe differences in performance for sequence tags and full-length reference sequences were surprisingly small. Characteristic variations in localization results for each program were noted that affect the localization of sequence at exon boundaries, in particular.