Dataset Information

Long-read amplicon denoising.

ABSTRACT: Long-read next-generation amplicon sequencing shows promise for studying complete genes or genomes from complex and diverse populations. Current long-read sequencing technologies have challenging error profiles, hindering data processing and incorporation into downstream analyses. Here we consider the problem of how to reconstruct, free of sequencing error, the true sequence variants and their associated frequencies from PacBio reads. Called 'amplicon denoising', this problem has been extensively studied for short-read sequencing technologies, but current solutions do not always successfully generalize to long reads with high indel error rates. We introduce two methods: one that runs nearly instantly and is very accurate for medium length reads and high template coverage, and another, slower method that is more robust when reads are very long or coverage is lower. On two Mock Virus Community datasets with ground truth, each sequenced on a different PacBio instrument, and on a number of simulated datasets, we compare our two approaches to each other and to existing algorithms. We outperform all tested methods in accuracy, with competitive run times even for our slower method, successfully discriminating templates that differ by a just single nucleotide. Julia implementations of Fast Amplicon Denoising (FAD) and Robust Amplicon Denoising (RAD), and a webserver interface, are freely available.

SUBMITTER: Kumar V

PROVIDER: S-EPMC6765106 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Long-read amplicon denoising.

Kumar Venkatesh V Vollbrecht Thomas T Chernyshev Mark M Mohan Sanjay S Hanst Brian B Bavafa Nicholas N Lorenzo Antonia A Kumar Nikesh N Ketteringham Robert R Eren Kemal K Golden Michael M Oliveira Michelli F MF Murrell Ben B

Nucleic acids research 20191001 18

Long-read next-generation amplicon sequencing shows promise for studying complete genes or genomes from complex and diverse populations. Current long-read sequencing technologies have challenging error profiles, hindering data processing and incorporation into downstream analyses. Here we consider the problem of how to reconstruct, free of sequencing error, the true sequence variants and their associated frequencies from PacBio reads. Called 'amplicon denoising', this problem has been extensivel ...[more]

PMID: 31418021

Similar Datasets

Project description:Reference sequence databases are critical to the accurate detection and identification of fungi in the environment. As repositories of large numbers of well-curated specimens, herbaria and fungal culture collections have the material resources to generate sequence data for large number of taxa, and could therefore allow filling taxonomic gaps often present in reference sequence databases. Financial resources to do that are however often lacking, so that recent efforts have focused on decreasing sequencing cost by increasing the number of multiplexed samples per sequencing run while maintaining high sequence quality. Following a previous study that aimed at decreasing sequencing cost for lichen specimens by generating fungal ITS barcodes for 96 specimens using PacBio amplicon sequencing, we present a method that further decreases lichen specimen metabarcoding costs. A total of 384 mixed DNA extracts obtained from lichen herbarium specimens, mostly from the four genera Buellia, Catillaria, Endocarpon and Parmotrema, were used to generate new fungal ITS sequences using a Sequel I sequencing platform and the PacBio M13 barcoded primers. The average success rate across all taxa was high (86.5%), with particularly high rates for the crustose saxicolous taxa (Buellia, Catillaria and others; 93.3%) and the terricolous squamulose taxa (Endocarpon and others; 96.5%). On the other hand, the success rate for the foliose genus Parmotrema was lower (60.4%). With this taxon sampling, greater specimen age did not appear to impact sequencing success. In fact, the 1966-1980 collection date category showed the highest success rate (97.3%). Compared to the previous study, the abundance-based sequence denoising method showed some limitations, but the cost of generating ITS barcodes was further decreased thanks to the higher multiplexing level. In addition to contributing new ITS barcodes for specimens of four interesting lichen genera, this study further highlights the potential and challenges of using new sequencing technologies on collection specimens to generate DNA sequences for reference databases.

Project description:BackgroundFreshwater ecosystems are inhabited by members of cosmopolitan bacterioplankton lineages despite the disconnected nature of these habitats. The lineages are delineated based on >?97% 16S rRNA gene sequence similarity, but their intra-lineage microdiversity and phylogeography, which are key to understanding the eco-evolutional processes behind their ubiquity, remain unresolved. Here, we applied long-read amplicon sequencing targeting nearly full-length 16S rRNA genes and the adjacent ribosomal internal transcribed spacer sequences to reveal the intra-lineage diversities of pelagic bacterioplankton assemblages in 11 deep freshwater lakes in Japan and Europe.ResultsOur single nucleotide-resolved analysis, which was validated using shotgun metagenomic sequencing, uncovered 7-101 amplicon sequence variants for each of the 11 predominant bacterial lineages and demonstrated sympatric, allopatric, and temporal microdiversities that could not be resolved through conventional approaches. Clusters of samples with similar intra-lineage population compositions were identified, which consistently supported genetic isolation between Japan and Europe. At a regional scale (up to hundreds of kilometers), dispersal between lakes was unlikely to be a limiting factor, and environmental factors or genetic drift were potential determinants of population composition. The extent of microdiversification varied among lineages, suggesting that highly diversified lineages (e.g., Iluma-A2 and acI-A1) achieve their ubiquity by containing a consortium of genotypes specific to each habitat, while less diversified lineages (e.g., CL500-11) may be ubiquitous due to a small number of widespread genotypes. The lowest extent of intra-lineage diversification was observed among the dominant hypolimnion-specific lineage (CL500-11), suggesting that their dispersal among lakes is not limited despite the hypolimnion being a more isolated habitat than the epilimnion.ConclusionsOur novel approach complemented the limited resolution of short-read amplicon sequencing and limited sensitivity of the metagenome assembly-based approach, and highlighted the complex ecological processes underlying the ubiquity of freshwater bacterioplankton lineages. To fully exploit the performance of the method, its relatively low read throughput is the major bottleneck to be overcome in the future. Video abstract.

Dataset Information

Long-read amplicon denoising.

Publications

Long-read amplicon denoising.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets