Nanopore ultra-long read sequencing technology for antimicrobial resistance detection in Mannheimia haemolytica

2019 ◽  
Vol 159 ◽  
pp. 138-147 ◽  
Author(s):  
Alexander Lim ◽  
Bryan Naidenov ◽  
Haley Bates ◽  
Karyn Willyerd ◽  
Timothy Snider ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Mannheimia Haemolytica ◽  
Sequencing Technology ◽  
Long Read ◽  
Resistance Detection

scholarly journals HapSolo: an optimization approach for removing secondary haplotigs during diploid genome assembly and scaffolding

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Edwin A. Solares ◽  
Yuan Tao ◽  
Anthony D. Long ◽  
Brandon S. Gaut
Keyword(s):  
Cost Function ◽  
Anopheles Funestus ◽  
Hill Climbing ◽  
Optimization Approach ◽  
Sequencing Technology ◽  
Genome Data ◽  
A Genome ◽  
Long Read ◽  
Downstream Analysis ◽  
The Cost

Abstract Background Despite marked recent improvements in long-read sequencing technology, the assembly of diploid genomes remains a difficult task. A major obstacle is distinguishing between alternative contigs that represent highly heterozygous regions. If primary and secondary contigs are not properly identified, the primary assembly will overrepresent both the size and complexity of the genome, which complicates downstream analysis such as scaffolding. Results Here we illustrate a new method, which we call HapSolo, that identifies secondary contigs and defines a primary assembly based on multiple pairwise contig alignment metrics. HapSolo evaluates candidate primary assemblies using BUSCO scores and then distinguishes among candidate assemblies using a cost function. The cost function can be defined by the user but by default considers the number of missing, duplicated and single BUSCO genes within the assembly. HapSolo performs hill climbing to minimize cost over thousands of candidate assemblies. We illustrate the performance of HapSolo on genome data from three species: the Chardonnay grape (Vitis vinifera), with a genome of 490 Mb, a mosquito (Anopheles funestus; 200 Mb) and the Thorny Skate (Amblyraja radiata; 2650 Mb). Conclusions HapSolo rapidly identified candidate assemblies that yield improvements in assembly metrics, including decreased genome size and improved N50 scores. Contig N50 scores improved by 35%, 9% and 9% for Chardonnay, mosquito and the thorny skate, respectively, relative to unreduced primary assemblies. The benefits of HapSolo were amplified by down-stream analyses, which we illustrated by scaffolding with Hi-C data. We found, for example, that prior to the application of HapSolo, only 52% of the Chardonnay genome was captured in the largest 19 scaffolds, corresponding to the number of chromosomes. After the application of HapSolo, this value increased to ~ 84%. The improvements for the mosquito’s largest three scaffolds, representing the number of chromosomes, were from 61 to 86%, and the improvement was even more pronounced for thorny skate. We compared the scaffolding results to assemblies that were based on PurgeDups for identifying secondary contigs, with generally superior results for HapSolo.

scholarly journals PacBio sequencing output increased through uniform and directional fivefold concatenation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nisha Kanwar ◽  
Celia Blanco ◽  
Irene A. Chen ◽  
Burckhard Seelig
Keyword(s):  
Improved Method ◽  
Sequencing Technology ◽  
Pacbio Sequencing ◽  
Limited Sample ◽  
Analysis Pipeline ◽  
Medium Length ◽  
Engineering Study ◽  
Long Read ◽  
The Cost ◽  
User Friendly

AbstractAdvances in sequencing technology have allowed researchers to sequence DNA with greater ease and at decreasing costs. Main developments have focused on either sequencing many short sequences or fewer large sequences. Methods for sequencing mid-sized sequences of 600–5,000 bp are currently less efficient. For example, the PacBio Sequel I system yields ~ 100,000–300,000 reads with an accuracy per base pair of 90–99%. We sought to sequence several DNA populations of ~ 870 bp in length with a sequencing accuracy of 99% and to the greatest depth possible. We optimised a simple, robust method to concatenate genes of ~ 870 bp five times and then sequenced the resulting DNA of ~ 5,000 bp by PacBioSMRT long-read sequencing. Our method improved upon previously published concatenation attempts, leading to a greater sequencing depth, high-quality reads and limited sample preparation at little expense. We applied this efficient concatenation protocol to sequence nine DNA populations from a protein engineering study. The improved method is accompanied by a simple and user-friendly analysis pipeline, DeCatCounter, to sequence medium-length sequences efficiently at one-fifth of the cost.

Serotyping and antimicrobial resistance of Mannheimia haemolytica strains from European cattle with bovine respiratory disease

2019 ◽  
Vol 124 ◽  
pp. 10-12 ◽  
Author(s):  
Sara Andrés-Lasheras ◽  
Rahat Zaheer ◽  
Cassidy Klima ◽  
Haley Sanderson ◽  
Rodrigo Ortega Polo ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Respiratory Disease ◽  
Bovine Respiratory Disease ◽  
Mannheimia Haemolytica ◽  
European Cattle

scholarly journals Defining Blood Group Gene Reference Alleles by Long-Read Sequencing: Proof of Concept in the ACKR1 Gene Encoding the Duffy Antigens

2019 ◽  
Vol 47 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Yann Fichou ◽  
Isabelle Berlivet ◽  
Gaëlle Richard ◽  
Christophe Tournamille ◽  
Lilian Castilho ◽  
...  
Keyword(s):  
Blood Group ◽  
Single Molecule ◽  
Pcr Amplification ◽  
Null Alleles ◽  
Sequencing Technology ◽  
Gene Encoding ◽  
Next Generation Sequencing Technology ◽  
Sequencing Technologies ◽  
Long Read ◽  
Long Range Pcr

Background: In the novel era of blood group genomics, (re-)defining reference gene/allele sequences of blood group genes has become an important goal to achieve, both for diagnostic and research purposes. As novel potent sequencing technologies are available, we thought to investigate the variability encountered in the three most common alleles of ACKR1, the gene encoding the clinically relevant Duffy antigens, at the haplotype level by a long-read sequencing approach. Materials and Methods: After long-range PCR amplification spanning the whole ACKR1 gene locus (∼2.5 kilobases), amplicons generated from 81 samples with known genotypes were sequenced in a single read by using the Pacific Biosciences (PacBio) single molecule, real-time (SMRT) sequencing technology. Results: High-quality sequencing reads were obtained for the 162 alleles (accuracy >0.999). Twenty-two nucleotide variations reported in databases were identified, defining 19 haplotypes: four, eight, and seven haplotypes in 46 ACKR1*01, 63 ACKR1*02, and 53 ACKR1*02N.01 alleles, respectively. Discussion: Overall, we have defined a subset of reference alleles by third-generation (long-read) sequencing. This technology, which provides a “longitudinal” overview of the loci of interest (several thousand base pairs) and is complementary to the second-generation (short-read) next-generation sequencing technology, is of critical interest for resolving novel, rare, and null alleles.

scholarly journals A recurrence-based approach for validating structural variation using long-read sequencing technology

GigaScience ◽  
2017 ◽  
Vol 6 (8) ◽  
Author(s):  
Xuefang Zhao ◽  
Alexandra M. Weber ◽  
Ryan E. Mills
Keyword(s):  
Structural Variation ◽  
Sequencing Technology ◽  
Long Read

scholarly journals Revealing complete complex KIR haplotypes phased by long-read sequencing technology

2017 ◽  
Vol 18 (3) ◽  
pp. 127-134 ◽  
Author(s):  
D Roe ◽  
C Vierra-Green ◽  
C-W Pyo ◽  
K Eng ◽  
R Hall ◽  
...  
Keyword(s):  
Sequencing Technology ◽  
Long Read ◽  
Kir Haplotypes

scholarly journals Differential overlap in human and animal fecal microbiomes and resistomes in rural versus urban Bangladesh

2021 ◽  
Author(s):  
Jenna M Swarthout ◽  
Erica R Fuhrmeister ◽  
Latifah Hamzah ◽  
Angela Harris ◽  
Mir A. Ahmed ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Urban Communities ◽  
Resistance Genes ◽  
Low Income ◽  
Urban Areas ◽  
Pathogenic Bacteria ◽  
Small Scale ◽  
Rrna Gene ◽  
Antimicrobial Resistance Genes ◽  
Long Read

Background Low- and middle-income countries (LMICs) bear the largest mortality burden due to antimicrobial-resistant infections. Small-scale animal production and free-roaming domestic animals are common in many LMICs, yet data on zoonotic exchange of gut bacteria and antimicrobial resistance genes (ARGs) in low-income communities are sparse. Differences between rural and urban communities in population density, antibiotic use, and cohabitation with animals likely influence the frequency of transmission of gut bacterial communities and ARGs between humans and animals. Here, we determined the similarity in gut microbiomes, using 16S rRNA gene amplicon sequencing, and resistomes, using long-read metagenomics, between humans, chickens, and goats in rural compared to urban Bangladesh. Results Gut microbiomes were more similar between humans and chickens in rural (where cohabitation is more common) compared to urban areas, but there was no difference for humans and goats. Urbanicity did not impact the similarity of human and animal resistomes; however, ARG abundance was higher in urban animals compared to rural animals. We identified substantial overlap of ARG alleles in humans and animals in both settings. Humans and chickens had more overlapping ARG alleles than humans and goats. All fecal hosts carried ARGs on contigs classified as potentially pathogenic bacteria, including Escherichia coli, Campylobacter jejuni, Clostridiodes difficile, and Klebsiella pneumoniae. Conclusions While the development of antimicrobial resistance in animal gut microbiomes and subsequent transmission to humans has been demonstrated in intensive farming environments and high-income countries, evidence of zoonotic exchange of antimicrobial resistance in LMIC communities is lacking. This research provides genomic evidence of overlap of antimicrobial resistance genes between humans and animals, especially in urban communities, and highlights chickens as important reservoirs of antimicrobial resistance. Chicken and human gut microbiomes were more similar in rural Bangladesh, where cohabitation is more common. Incorporation of long-read metagenomics enabled characterization of bacterial hosts of resistance genes, which has not been possible in previous culture-independent studies using only short-read sequencing. These findings highlight the importance of developing strategies for combatting antimicrobial resistance that account for chickens being reservoirs of ARGs in community environments, especially in urban areas.

scholarly journals QAlign: Aligning nanopore reads accurately using current-level modeling

2019 ◽  
Author(s):  
Dhaivat Joshi ◽  
Shunfu Mao ◽  
Sreeram Kannan ◽  
Suhas Diggavi
Keyword(s):  
Reference Genome ◽  
Genomic Analysis ◽  
Vital Role ◽  
High Error Rate ◽  
Sequencing Technology ◽  
Long Reads ◽  
A Genome ◽  
Long Read ◽  
Nanopore Sequencer ◽  
Sequencing Process

AbstractMotivationEfficient and accurate alignment of DNA / RNA sequence reads to each other or to a reference genome / transcriptome is an important problem in genomic analysis. Nanopore sequencing has emerged as a major sequencing technology and many long-read aligners have been designed for aligning nanopore reads. However, the high error rate makes accurate and efficient alignment difficult. Utilizing the noise and error characteristics inherent in the sequencing process properly can play a vital role in constructing a robust aligner. In this paper, we design QAlign, a pre-processor that can be used with any long-read aligner for aligning long reads to a genome / transcriptome or to other long reads. The key idea in QAlign is to convert the nucleotide reads into discretized current levels that capture the error modes of the nanopore sequencer before running it through a sequence aligner.ResultsWe show that QAlign is able to improve alignment rates from around 80% up to 90% with nanopore reads when aligning to the genome. We also show that QAlign improves the average overlap quality by 9.2%, 2.5% and 10.8% in three real datasets for read-to-read alignment. Read-to-transcriptome alignment rates are improved from 51.6% to 75.4% and 82.6% to 90% in two real datasets.Availabilityhttps://github.com/joshidhaivat/QAlign.git

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