Menu

Asset Tag: Barcoding

July 7, 2019  |  

Advances in DNA sequencing technologies for high resolution HLA typing.

This communication describes our experience in large-scale G group-level high resolution HLA typing using three different DNA sequencing platforms – ABI 3730 xl, Illumina MiSeq and PacBio RS II. Recent advances in DNA sequencing technologies, so-called next generation sequencing (NGS), have brought breakthroughs in deciphering the genetic information in all living species at a large scale and at an affordable level. The NGS DNA indexing system allows sequencing multiple genes for large number of individuals in a single run. Our laboratory has adopted and used these technologies for HLA molecular testing services. We found that each sequencing technology has its own strengths and weaknesses, and their sequencing performances complement each other. HLA genes are highly complex and genotyping them is quite challenging. Using these three sequencing platforms, we were able to meet all requirements for G group-level high resolution and high volume HLA typing. Copyright © 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Read more
July 7, 2019  |  

Enhancing the detection of barcoded reads in high throughput DNA sequencing data by controlling the false discovery rate.

DNA barcodes are short unique sequences used to label DNA or RNA-derived samples in multiplexed deep sequencing experiments. During the demultiplexing step, barcodes must be detected and their position identified. In some cases (e.g., with PacBio SMRT), the position of the barcode and DNA context is not well defined. Many reads start inside the genomic insert so that adjacent primers might be missed. The matter is further complicated by coincidental similarities between barcode sequences and reference DNA. Therefore, a robust strategy is required in order to detect barcoded reads and avoid a large number of false positives or negatives.For mass inference problems such as this one, false discovery rate (FDR) methods are powerful and balanced solutions. Since existing FDR methods cannot be applied to this particular problem, we present an adapted FDR method that is suitable for the detection of barcoded reads as well as suggest possible improvements.In our analysis, barcode sequences showed high rates of coincidental similarities with the Mus musculus reference DNA. This problem became more acute when the length of the barcode sequence decreased and the number of barcodes in the set increased. The method presented in this paper controls the tail area-based false discovery rate to distinguish between barcoded and unbarcoded reads. This method helps to establish the highest acceptable minimal distance between reads and barcode sequences. In a proof of concept experiment we correctly detected barcodes in 83% of the reads with a precision of 89%. Sensitivity improved to 99% at 99% precision when the adjacent primer sequence was incorporated in the analysis. The analysis was further improved using a paired end strategy. Following an analysis of the data for sequence variants induced in the Atp1a1 gene of C57BL/6 murine melanocytes by ultraviolet light and conferring resistance to ouabain, we found no evidence of cross-contamination of DNA material between samples.Our method offers a proper quantitative treatment of the problem of detecting barcoded reads in a noisy sequencing environment. It is based on the false discovery rate statistics that allows a proper trade-off between sensitivity and precision to be chosen.

Read more
July 7, 2019  |  

Next-generation polyploid phylogenetics: rapid resolution of hybrid polyploid complexes using PacBio single-molecule sequencing.

Difficulties in generating nuclear data for polyploids have impeded phylogenetic study of these groups. We describe a high-throughput protocol and an associated bioinformatics pipeline (Pipeline for Untangling Reticulate Complexes (Purc)) that is able to generate these data quickly and conveniently, and demonstrate its efficacy on accessions from the fern family Cystopteridaceae. We conclude with a demonstration of the downstream utility of these data by inferring a multi-labeled species tree for a subset of our accessions. We amplified four c. 1-kb-long nuclear loci and sequenced them in a parallel-tagged amplicon sequencing approach using the PacBio platform. Purc infers the final sequences from the raw reads via an iterative approach that corrects PCR and sequencing errors and removes PCR-mediated recombinant sequences (chimeras). We generated data for all gene copies (homeologs, paralogs, and segregating alleles) present in each of three sets of 50 mostly polyploid accessions, for four loci, in three PacBio runs (one run per set). From the raw sequencing reads, Purc was able to accurately infer the underlying sequences. This approach makes it easy and economical to study the phylogenetics of polyploids, and, in conjunction with recent analytical advances, facilitates investigation of broad patterns of polyploid evolution.© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Read more
July 7, 2019  |  

Prevalence and molecular characterization of mcr-1-positive Salmonella strains recovered from clinical specimens in China.

The recently discovered colistin resistance element, mcr-1, adds to the list of antimicrobial resistance genes that rapidly erode the antimicrobial efficacy of not only the commonly used antibiotics but also the last-line agents of carbapenems and colistin. This study investigated the prevalence of the mobile colistin resistance determinant mcr-1 in Salmonella strains recovered from clinical settings in China and the transmission potential of mcr-1-bearing mobile elements harbored by such isolates. The mcr-1 gene was recoverable in 1.4% of clinical isolates tested, with the majority of them belonging to Salmonella enterica serotype Typhimurium. These isolates exhibited diverse pulsed-field gel electrophoresis (PFGE) profiles and high resistance to antibiotics other than colistin and particularly to cephalosporins. Plasmid analysis showed that mcr-1 was carried on a variety of plasmids with sizes ranging from ~30 to ~250 kb, among which there were conjugative plasmids of ~30 kb, ~60 kb, and ~250 kb and nonconjugative plasmids of ~140 kb, ~180 kb, and ~240 kb. Sequencing of representative mcr-1-carrying plasmids revealed that all conjugative plasmids belonged to the IncX4, IncI2, and IncHI2 types and were highly similar to the corresponding types of plasmids reported previously. Nonconjugative plasmids all belonged to the IncHI2 type, and the nontransferability of these plasmids was attributed to the loss of a region carrying partial or complete tra genes. Our data revealed that, similar to the situation in Escherichia coli, mcr-1 transmission in Salmonella was accelerated by various plasmids, suggesting that transmission of mcr-1-carrying plasmids between different species of Enterobacteriaceae may be a common event. Copyright © 2017 American Society for Microbiology.

Read more
July 7, 2019  |  

Mistranslation can enhance fitness through purging of deleterious mutations.

Phenotypic mutations are amino acid changes caused by mistranslation. How phenotypic mutations affect the adaptive evolution of new protein functions is unknown. Here we evolve the antibiotic resistance protein TEM-1 towards resistance on the antibiotic cefotaxime in an Escherichia coli strain with a high mistranslation rate. TEM-1 populations evolved in such strains endow host cells with a general growth advantage, not only on cefotaxime but also on several other antibiotics that ancestral TEM-1 had been unable to deactivate. High-throughput sequencing of TEM-1 populations shows that this advantage is associated with a lower incidence of weakly deleterious genotypic mutations. Our observations show that mistranslation is not just a source of noise that delays adaptive evolution. It could even facilitate adaptive evolution by exacerbating the effects of deleterious mutations and leading to their more efficient purging. The ubiquity of mistranslation and its effects render mistranslation an important factor in adaptive protein evolution.

Read more
July 7, 2019  |  

Complete genome of a panresistant Pseudomonas aeruginosa strain, isolated from a patient with respiratory failure in a Canadian community hospital.

We report here the complete genome sequence of a panresistant Pseudomonas aeruginosa strain, isolated from a patient with respiratory failure in Canada. No carbapenemase genes were identified. Carbapenem resistance is attributable to a frameshift in the oprD gene; the basis for colistin resistance remains undetermined. Copyright © 2017 Xiong et al.

Read more
July 7, 2019  |  

Evolutionary origin of the staphylococcal cassette chromosome mec (SCCmec).

Several lines of evidence indicate that the most primitive staphylococcal species, those of the Staphylococcus sciuri group, were involved in the first stages of evolution of the staphylococcal cassette chromosome mec (SCCmec), the genetic element carrying the ß-lactam resistance gene mecA However, many steps are still missing from this evolutionary history. In particular, it is not known how mecA was incorporated into the mobile element SCC prior to dissemination among Staphylococcus aureus and other pathogenic staphylococcal species. To gain insights into the possible contribution of several species of the Staphylococcus sciuri group to the assembly of SCCmec, we sequenced the genomes of 106 isolates, comprising S. sciuri (n = 76), Staphylococcus vitulinus (n = 18), and Staphylococcus fleurettii (n = 12) from animal and human sources, and characterized the native location of mecA and the SCC insertion site by using a variety of comparative genomic approaches. Moreover, we performed a single nucleotide polymorphism (SNP) analysis of the genomes in order to understand SCCmec evolution in relation to phylogeny. We found that each of three species of the S. sciuri group contributed to the evolution of SCCmec: S. vitulinus and S. fleurettii contributed to the assembly of the mec complex, and S. sciuri most likely provided the mobile element in which mecA was later incorporated. We hypothesize that an ancestral SCCmec III cassette (an element carried by one of the most epidemic methicillin-resistant S. aureus clones) originated in S. sciuri possibly by a recombination event in a human host or a human-created environment and later was transferred to S. aureus. Copyright © 2017 American Society for Microbiology.

Read more
July 7, 2019  |  

MHC class I diversity in chimpanzees and bonobos.

Major histocompatibility complex (MHC) class I genes are critically involved in the defense against intracellular pathogens. MHC diversity comparisons among samples of closely related taxa may reveal traces of past or ongoing selective processes. The bonobo and chimpanzee are the closest living evolutionary relatives of humans and last shared a common ancestor some 1 mya. However, little is known concerning MHC class I diversity in bonobos or in central chimpanzees, the most numerous and genetically diverse chimpanzee subspecies. Here, we used a long-read sequencing technology (PacBio) to sequence the classical MHC class I genes A, B, C, and A-like in 20 and 30 wild-born bonobos and chimpanzees, respectively, with a main focus on central chimpanzees to assess and compare diversity in those two species. We describe in total 21 and 42 novel coding region sequences for the two species, respectively. In addition, we found evidence for a reduced MHC class I diversity in bonobos as compared to central chimpanzees as well as to western chimpanzees and humans. The reduced bonobo MHC class I diversity may be the result of a selective process in their evolutionary past since their split from chimpanzees.

Read more
July 7, 2019  |  

Designing robust watermark barcodes for multiplex long-read sequencing.

To attain acceptable sample misassignment rates, current approaches to multiplex single-molecule real-time sequencing require upstream quality improvement, which is obtained from multiple passes over the sequenced insert and significantly reduces the effective read length. In order to fully exploit the raw read length on multiplex applications, robust barcodes capable of dealing with the full single-pass error rates are needed.We present a method for designing sequencing barcodes that can withstand a large number of insertion, deletion and substitution errors and are suitable for use in multiplex single-molecule real-time sequencing. The manuscript focuses on the design of barcodes for full-length single-pass reads, impaired by challenging error rates in the order of 11%. The proposed barcodes can multiplex hundreds or thousands of samples while achieving sample misassignment probabilities as low as 10-7 under the above conditions, and are designed to be compatible with chemical constraints imposed by the sequencing process.Software tools for constructing watermark barcode sets and demultiplexing barcoded reads, together with example sets of barcodes and synthetic barcoded reads, are freely available at www.cifasis-conicet.gov.ar/ezpeleta/NS-watermark .ezpeleta@cifasis-conicet.gov.ar.

Read more
July 7, 2019  |  

Generation of a collection of mutant tomato lines using pooled CRISPR libraries.

The high efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)-mediated mutagenesis in plants enables the development of high-throughput mutagenesis strategies. By transforming pooled CRISPR libraries into tomato (Solanum lycopersicum), collections of mutant lines were generated with minimal transformation attempts and in a relatively short period of time. Identification of the targeted gene(s) was easily determined by sequencing the incorporated guide RNA(s) in the primary transgenic events. From a single transformation with a CRISPR library targeting the immunity-associated leucine-rich repeat subfamily XII genes, heritable mutations were recovered in 15 of the 54 genes targeted. To increase throughput, a second CRISPR library was made containing three guide RNAs per construct to target 18 putative transporter genes. This resulted in stable mutations in 15 of the 18 targeted genes, with some primary transgenic plants having as many as five mutated genes. Furthermore, the redundancy in this collection of plants allowed for the association of aberrant T0 phenotypes with the underlying targeted genes. Plants with mutations in a homolog of an Arabidopsis (Arabidopsis thaliana) boron efflux transporter displayed boron deficiency phenotypes. The strategy described here provides a technically simple yet high-throughput approach for generating a collection of lines with targeted mutations and should be applicable to any plant transformation system.© 2017 American Society of Plant Biologists. All Rights Reserved.

Read more
July 7, 2019  |  

Archetype JC polyomavirus prevails in a rare case of JC polyomavirus nephropathy and in stable renal transplant recipients with JC polyomavirus viruria.

JC polyomavirus (JCPyV) is reactivated in approximately 20% of renal transplant recipients and it may rarely cause JCPyV-associated nephropathy (JCPyVAN). Whereas progressive multifocal leukoencephalopathy of the brain is caused by rearranged neurotropic JCPyV, little is known about viral sequence variation in JCPyVAN due to the rarity of this condition.Using single-molecule real-time sequencing, characterization of full-length JCPyV genomes from urine and plasma of one JCPyVAN patient and twenty stable renal transplant recipients with JCPyV viruria was attempted. Sequence analysis of JCPyV strains was performed with the emphasis on the NCCR region, the major capsid protein gene VP1 and the large T antigen (LTag) gene.Exclusively archetype strains were identified in urine of the JCPyVAN patient. Full-length JCPyV sequences were not retrieved from plasma. Archetype strains were found in urine of nineteen stable renal transplant recipients, with JCPyV quasispecies detected in five samples. In a patient with minor graft dysfunction, a strain with archetype-like NCCR region was discovered. Individual point mutations were detected in both VP1 and LTag genes.Archetype JCPyV was dominant in the JCPyVAN patient and in stable renal transplant recipients. Archetype rather than rearranged JCPyV seems to drive the pathogenesis of JCPyVAN.

Read more
July 7, 2019  |  

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.© The Authors 2017. Published by Oxford University Press.

Read more
July 7, 2019  |  

Genomic patterns of de novo mutation in simplex autism.

To further our understanding of the genetic etiology of autism, we generated and analyzed genome sequence data from 516 idiopathic autism families (2,064 individuals). This resource includes >59 million single-nucleotide variants (SNVs) and 9,212 private copy number variants (CNVs), of which 133,992 and 88 are de novo mutations (DNMs), respectively. We estimate a mutation rate of ~1.5 × 10(-8) SNVs per site per generation with a significantly higher mutation rate in repetitive DNA. Comparing probands and unaffected siblings, we observe several DNM trends. Probands carry more gene-disruptive CNVs and SNVs, resulting in severe missense mutations and mapping to predicted fetal brain promoters and embryonic stem cell enhancers. These differences become more pronounced for autism genes (p = 1.8 × 10(-3), OR = 2.2). Patients are more likely to carry multiple coding and noncoding DNMs in different genes, which are enriched for expression in striatal neurons (p = 3 × 10(-3)), suggesting a path forward for genetically characterizing more complex cases of autism. Copyright © 2017 Elsevier Inc. All rights reserved.

Read more
July 7, 2019  |  

A new species of Xenoturbella from the western Pacific Ocean and the evolution of Xenoturbella.

Xenoturbella is a group of marine benthic animals lacking an anus and a centralized nervous system. Molecular phylogenetic analyses group the animal together with the Acoelomorpha, forming the Xenacoelomorpha. This group has been suggested to be either a sister group to the Nephrozoa or a deuterostome, and therefore it may provide important insights into origins of bilaterian traits such as an anus, the nephron, feeding larvae and centralized nervous systems. However, only five Xenoturbella species have been reported and the evolutionary history of xenoturbellids and Xenacoelomorpha remains obscure.Here we describe a new Xenoturbella species from the western Pacific Ocean, and report a new xenoturbellid structure – the frontal pore. Non-destructive microCT was used to investigate the internal morphology of this soft-bodied animal. This revealed the presence of a frontal pore that is continuous with the ventral glandular network and which exhibits similarities with the frontal organ in acoelomorphs.Our results suggest that large size, oval mouth, frontal pore and ventral glandular network may be ancestral features for Xenoturbella. Further studies will clarify the evolutionary relationship of the frontal pore and ventral glandular network of xenoturbellids and the acoelomorph frontal organ. One of the habitats of the newly identified species is easily accessible from a marine station and so this species promises to be valuable for research on bilaterian and deuterostome evolution.

Read more
July 7, 2019  |  

Environmental changes bridge evolutionary valleys.

In the basic fitness landscape metaphor for molecular evolution, evolutionary pathways are presumed to follow uphill steps of increasing fitness. How evolution can cross fitness valleys is an open question. One possibility is that environmental changes alter the fitness landscape such that low-fitness sequences reside on a hill in alternate environments. We experimentally test this hypothesis on the antibiotic resistance gene TEM-15 ß-lactamase by comparing four evolutionary strategies shaped by environmental changes. The strategy that included initial steps of selecting for low antibiotic resistance (negative selection) produced superior alleles compared with the other three strategies. We comprehensively examined possible evolutionary pathways leading to one such high-fitness allele and found that an initially deleterious mutation is key to the allele’s evolutionary history. This mutation is an initial gateway to an otherwise relatively inaccessible area of sequence space and participates in higher-order, positive epistasis with a number of neutral to slightly beneficial mutations. The ability of negative selection and environmental changes to provide access to novel fitness peaks has important implications for natural evolutionary mechanisms and applied directed evolution.

Read more

Subscribe for blog updates:

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.