Genomic outcomes from diverse SNP panels obtained with different de novo building-loci pipelines in a varied species context: Can pipelines be influencing biological interpretations?

Abstract Background: The irruption of Next-generation sequencing (NGS) and restriction site-associated DNA sequencing (RAD-seq) in the last decade has led to the identification of thousands of molecular markers and their genotyping for refined genomic screening. This approach has been especially useful for non-model organisms with limited genomic resources. Many building-loci pipelines have been developed to obtain robust single nucleotide polymorphism (SNPs) genotyping datasets using a de novo RAD-seq approach, i.e. without reference genomes. Here, the performances of two building-loci pipelines, STACKS 2 and Meyer’s 2b-RAD v2.1 pipeline, were compared using a diverse set of aquatic species representing different genomic and/or population structure scenarios. Two bivalve species (Manila clam and common edible cockle) and three fish species (brown trout, silver catfish and small-spotted catshark) were studied. Four SNP panels were evaluated in each species to test both different building-loci pipelines and criteria for SNP selection. Furthermore, for Manila clam and brown trout, a reference genome approach was used as control. Results: Despite different outcomes were observed between pipelines and species with the diverse SNP calling and filtering steps tested, no remarkable differences were found on genetic diversity and differentiation within species with the SNP panels obtained with a de novo approach. The main differences were found in brown trout between the de novo and reference genome approaches. Genotyped vs missing data mismatches were the main genotyping difference detected between the two building-loci pipelines or between the de novo and reference genome comparisons. Conclusions: Tested building-loci pipelines seem not to have a substantial influence on population genetics inference. Preliminary trials with bioinformatic pipelines are suggested to evaluate their influence in population parameters related to the specific goals of the study.

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Genomic outcomes from diverse SNP panels obtained with different de novo building-loci pipelines in a varied species context: Can pipelines be influencing biological interpretations?

10.21203/rs.3.rs-50690/v1 ◽

2020 ◽

Author(s):

Adrian Casanova ◽

Francesco Maroso ◽

Andrés Blanco ◽

Miguel Hermida ◽

Nestor Rios ◽

...

Keyword(s):

Genetic Diversity ◽

Brown Trout ◽

Reference Genome ◽

De Novo ◽

Manila Clam ◽

Model Organisms ◽

Single Nucleotide ◽

Snp Panels ◽

Next Generation Sequencing Ngs ◽

Genome Comparisons

Abstract Background The irruption of Next-generation sequencing (NGS) and restriction site-associated DNA sequencing (RAD-seq) in the last decade has led to the identification of thousands of molecular markers and their genotyping for refined genomic screening. This approach has been especially useful for non-model organisms with limited genomic resources. Many building-loci pipelines have been developed to obtain robust single nucleotide polymorphism (SNPs) genotyping datasets using a de novo RAD-seq approach, i.e. without reference genomes. Here, the performances of two building-loci pipelines, STACKS 2 and Meyer’s 2b-RAD v2.1 pipeline, were compared using a diverse set of aquatic species representing different genomic and/or population structure scenarios. Two bivalve species (Manila clam and common edible cockle) and three fish species (brown trout, silver catfish and small-spotted catshark) were studied. Four SNP panels were evaluated in each species to test both different building-loci pipelines and criteria for SNP selection. Furthermore, for Manila clam and brown trout, a reference genome approach was used as control. Results Despite different outcomes were observed between pipelines and species with the diverse SNP calling and filtering steps tested, no remarkable differences were found on genetic diversity and differentiation within species with the SNP panels obtained with a de novo approach. The main differences were found in brown trout between the de novo and reference genome approaches. Genotyped vs missing data mismatches were the main genotyping difference detected between the two building-loci pipelines or between the de novo and reference genome comparisons. Conclusions Building-loci pipelines seem not to have a substantial influence on population genetics inference. Anyway, we recommend being careful with certain building-loci pipeline parameters and SNP filtering steps, especially when a de novo approach is used. Preliminary trials with subsets of data should be performed for comparison of genetic diversity and differentiation, but always considering the specific goals of the study.

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Low impact of different SNP panels from two building-loci pipelines on RAD-Seq population genomic metrics: case study on five diverse aquatic species

BMC Genomics ◽

10.1186/s12864-021-07465-w ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Adrián Casanova ◽

Francesco Maroso ◽

Andrés Blanco ◽

Miguel Hermida ◽

Néstor Ríos ◽

...

Keyword(s):

Brown Trout ◽

Reference Genome ◽

De Novo ◽

Manila Clam ◽

Model Organisms ◽

Aquatic Species ◽

Single Nucleotide ◽

Snp Panels ◽

Next Generation Sequencing Ngs

Abstract Background The irruption of Next-generation sequencing (NGS) and restriction site-associated DNA sequencing (RAD-seq) in the last decade has led to the identification of thousands of molecular markers and their genotyping for refined genomic screening. This approach has been especially useful for non-model organisms with limited genomic resources. Many building-loci pipelines have been developed to obtain robust single nucleotide polymorphism (SNPs) genotyping datasets using a de novo RAD-seq approach, i.e. without reference genomes. Here, the performances of two building-loci pipelines, STACKS 2 and Meyer’s 2b-RAD v2.1 pipeline, were compared using a diverse set of aquatic species representing different genomic and/or population structure scenarios. Two bivalve species (Manila clam and common edible cockle) and three fish species (brown trout, silver catfish and small-spotted catshark) were studied. Four SNP panels were evaluated in each species to test both different building-loci pipelines and criteria for SNP selection. Furthermore, for Manila clam and brown trout, a reference genome approach was used as control. Results Despite different outcomes were observed between pipelines and species with the diverse SNP calling and filtering steps tested, no remarkable differences were found on genetic diversity and differentiation within species with the SNP panels obtained with a de novo approach. The main differences were found in brown trout between the de novo and reference genome approaches. Genotyped vs missing data mismatches were the main genotyping difference detected between the two building-loci pipelines or between the de novo and reference genome comparisons. Conclusions Tested building-loci pipelines for selection of SNP panels seem to have low influence on population genetics inference across the diverse case-study scenarios here studied. However, preliminary trials with different bioinformatic pipelines are suggested to evaluate their influence on population parameters according with the specific goals of each study.

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AStrap: identification of alternative splicing from transcript sequences without a reference genome

Bioinformatics ◽

10.1093/bioinformatics/bty1008 ◽

2018 ◽

Vol 35 (15) ◽

pp. 2654-2656 ◽

Cited By ~ 5

Author(s):

Guoli Ji ◽

Wenbin Ye ◽

Yaru Su ◽

Moliang Chen ◽

Guangzao Huang ◽

...

Keyword(s):

Machine Learning ◽

Alternative Splicing ◽

Single Molecule ◽

Reference Genome ◽

De Novo ◽

Supplementary Information ◽

Model Organisms ◽

Sequencing Data ◽

Extensive Evaluation ◽

Reference Genomes

Abstract Summary Alternative splicing (AS) is a well-established mechanism for increasing transcriptome and proteome diversity, however, detecting AS events and distinguishing among AS types in organisms without available reference genomes remains challenging. We developed a de novo approach called AStrap for AS analysis without using a reference genome. AStrap identifies AS events by extensive pair-wise alignments of transcript sequences and predicts AS types by a machine-learning model integrating more than 500 assembled features. We evaluated AStrap using collected AS events from reference genomes of rice and human as well as single-molecule real-time sequencing data from Amborella trichopoda. Results show that AStrap can identify much more AS events with comparable or higher accuracy than the competing method. AStrap also possesses a unique feature of predicting AS types, which achieves an overall accuracy of ∼0.87 for different species. Extensive evaluation of AStrap using different parameters, sample sizes and machine-learning models on different species also demonstrates the robustness and flexibility of AStrap. AStrap could be a valuable addition to the community for the study of AS in non-model organisms with limited genetic resources. Availability and implementation AStrap is available for download at https://github.com/BMILAB/AStrap. Supplementary information Supplementary data are available at Bioinformatics online.

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DiscoSnp++: de novo detection of small variants from raw unassembled read set(s)

10.1101/209965 ◽

2017 ◽

Cited By ~ 12

Author(s):

Pierre Peterlongo ◽

Chloé Riou ◽

Erwan Drezen ◽

Claire Lemaitre

Keyword(s):

Reference Genome ◽

De Novo ◽

Model Organisms ◽

Small Indels ◽

Desktop Computers ◽

Resource Requirements ◽

Computational Resources ◽

Next Generation Sequencing Ngs ◽

Ngs Data ◽

Source Of Information

AbstractMotivationNext Generation Sequencing (NGS) data provide an unprecedented access to life mechanisms. In particular, these data enable to detect polymorphisms such as SNPs and indels. As these polymorphisms represent a fundamental source of information in agronomy, environment or medicine, their detection in NGS data is now a routine task. The main methods for their prediction usually need a reference genome. However, non-model organisms and highly divergent genomes such as in cancer studies are extensively investigated.ResultsWe propose DiscoSnp++, in which we revisit the DiscoSnp algorithm. DiscoSnp++ is designed for detecting and ranking all kinds of SNPs and small indels from raw read set(s). It outputs files in fasta and VCF formats. In particular, predicted variants can be automatically localized afterwards on a reference genome if available. Its usage is extremely simple and its low resource requirements make it usable on common desktop computers. Results show that DiscoSnp++ performs better than state-of-the-art methods in terms of computational resources and in terms of results quality. An important novelty is the de novo detection of indels, for which we obtained 99% precision when calling indels on simulated human datasets and 90% recall on high confident indels from the Platinum dataset.LicenseGNU Affero general public licenseAvailabilityhttps://github.com/GATB/[email protected]

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SuperTranscript: a data driven reference for analysis and visualisation of transcriptomes

10.1101/077750 ◽

2016 ◽

Cited By ~ 3

Author(s):

Nadia M Davidson ◽

Anthony DK Hawkins ◽

Alicia Oshlack

Keyword(s):

Reference Genome ◽

De Novo ◽

Transcriptome Assembly ◽

Model Organisms ◽

Sequencing Data ◽

Expressed Sequence ◽

Model Sequencing ◽

Reference Genomes ◽

Exonic Sequence ◽

Single Sequence

AbstractNumerous methods have been developed to analyse RNA sequencing data, but most rely on the availability of a reference genome, making them unsuitable for non-model organisms. De novo transcriptome assembly can build a reference transcriptome from the non-model sequencing data, but falls short of allowing most tools to be applied. Here we present superTranscripts, a simple but powerful solution to bridge that gap. SuperTranscripts are a substitute for a reference genome, consisting of all the unique exonic sequence, in transcriptional order, such that each gene is represented by a single sequence. We demonstrate how superTranscripts allow visualization, variant detection and differential isoform detection in non-model organisms, using widely applied methods that are designed to work with reference genomes. SuperTranscripts can also be applied to model organisms to enhance visualization and discover novel expressed sequence. We describe Lace, software to construct superTranscripts from any set of transcripts including de novo assembled transcriptomes. In addition we used Lace to combine reference and assembled transcriptomes for chicken and recovered the sequence of hundreds of gaps in the reference genome.

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In Search of Species-Specific SNPs in a Non-Model Animal (European Bison (Bison bonasus))—Comparison of De Novo and Reference-Based Integrated Pipeline of STACKS Using Genotyping-by-Sequencing (GBS) Data

Animals ◽

10.3390/ani11082226 ◽

2021 ◽

Vol 11 (8) ◽

pp. 2226

Author(s):

Sazia Kunvar ◽

Sylwia Czarnomska ◽

Cino Pertoldi ◽

Małgorzata Tokarska

Keyword(s):

Reference Genome ◽

De Novo ◽

Bos Taurus ◽

Model Organism ◽

Genotyping By Sequencing ◽

Model Organisms ◽

European Bison ◽

Model Animal ◽

Pcr Duplicates ◽

Species Specific

The European bison is a non-model organism; thus, most of its genetic and genomic analyses have been performed using cattle-specific resources, such as BovineSNP50 BeadChip or Illumina Bovine 800 K HD Bead Chip. The problem with non-specific tools is the potential loss of evolutionary diversified information (ascertainment bias) and species-specific markers. Here, we have used a genotyping-by-sequencing (GBS) approach for genotyping 256 samples from the European bison population in Bialowieza Forest (Poland) and performed an analysis using two integrated pipelines of the STACKS software: one is de novo (without reference genome) and the other is a reference pipeline (with reference genome). Moreover, we used a reference pipeline with two different genomes, i.e., Bos taurus and European bison. Genotyping by sequencing (GBS) is a useful tool for SNP genotyping in non-model organisms due to its cost effectiveness. Our results support GBS with a reference pipeline without PCR duplicates as a powerful approach for studying the population structure and genotyping data of non-model organisms. We found more polymorphic markers in the reference pipeline in comparison to the de novo pipeline. The decreased number of SNPs from the de novo pipeline could be due to the extremely low level of heterozygosity in European bison. It has been confirmed that all the de novo/Bos taurus and Bos taurus reference pipeline obtained SNPs were unique and not included in 800 K BovineHD BeadChip.

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benchNGS : An approach to benchmark short reads alignment tools

10.7287/peerj.preprints.1007v1 ◽

2015 ◽

Author(s):

Farzana Rahman ◽

Mehedi Hassan ◽

Alona Kryshchenko ◽

Inna Dubchak ◽

Tatiana V Tatarinova ◽

...

Keyword(s):

Reference Genome ◽

Global Alignment ◽

Short Report ◽

Related Genome ◽

Genome Sequences ◽

Short Reads ◽

Relevant Reference ◽

Next Generation Sequencing Ngs ◽

Reference Genomes ◽

Generation Sequencing

In the last decade a number of algorithms and associated software were developed to align next generation sequencing (NGS) reads to relevant reference genomes. The results of these programs may vary significantly, especially when the NGS reads are contain mutations not found in the reference genome. Yet there is no standard way to compare these programs and assess their biological relevance. We propose a benchmark to assess accuracy of the short reads mapping based on the pre-computed global alignment of closely related genome sequences. In this paper we outline the method and also present a short report of an experiment performed on five popular alignment tools .

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dnAQET: a framework to compute a consolidated metric for benchmarking quality of de novo assemblies

BMC Genomics ◽

10.1186/s12864-019-6070-x ◽

2019 ◽

Vol 20 (1) ◽

Author(s):

Gokhan Yavas ◽

Huixiao Hong ◽

Wenming Xiao

Keyword(s):

Quality Assessment ◽

Genome Assembly ◽

Reference Genome ◽

De Novo ◽

Quality Score ◽

De Novo Genome Assembly ◽

Genome Assemblies ◽

Reference Genomes ◽

Better Than

Abstract Background Accurate de novo genome assembly has become reality with the advancements in sequencing technology. With the ever-increasing number of de novo genome assembly tools, assessing the quality of assemblies has become of great importance in genome research. Although many quality metrics have been proposed and software tools for calculating those metrics have been developed, the existing tools do not produce a unified measure to reflect the overall quality of an assembly. Results To address this issue, we developed the de novo Assembly Quality Evaluation Tool (dnAQET) that generates a unified metric for benchmarking the quality assessment of assemblies. Our framework first calculates individual quality scores for the scaffolds/contigs of an assembly by aligning them to a reference genome. Next, it computes a quality score for the assembly using its overall reference genome coverage, the quality score distribution of its scaffolds and the redundancy identified in it. Using synthetic assemblies randomly generated from the latest human genome build, various builds of the reference genomes for five organisms and six de novo assemblies for sample NA24385, we tested dnAQET to assess its capability for benchmarking quality evaluation of genome assemblies. For synthetic data, our quality score increased with decreasing number of misassemblies and redundancy and increasing average contig length and coverage, as expected. For genome builds, dnAQET quality score calculated for a more recent reference genome was better than the score for an older version. To compare with some of the most frequently used measures, 13 other quality measures were calculated. The quality score from dnAQET was found to be better than all other measures in terms of consistency with the known quality of the reference genomes, indicating that dnAQET is reliable for benchmarking quality assessment of de novo genome assemblies. Conclusions The dnAQET is a scalable framework designed to evaluate a de novo genome assembly based on the aggregated quality of its scaffolds (or contigs). Our results demonstrated that dnAQET quality score is reliable for benchmarking quality assessment of genome assemblies. The dnQAET can help researchers to identify the most suitable assembly tools and to select high quality assemblies generated.

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SMRT Genome Assembly Corrects Reference Errors, Resolving the Genetic Basis of Virulence in Mycobacterium tuberculosis

10.1101/064840 ◽

2016 ◽

Author(s):

Afif Elghraoui ◽

Samuel J Modlin ◽

Faramarz Valafar

Keyword(s):

Mycobacterium Tuberculosis ◽

Single Molecule ◽

Genetic Basis ◽

Reference Genome ◽

Reference Sequence ◽

Smrt Sequencing ◽

Virulence Attenuation ◽

Sequencing Platforms ◽

Genome Comparisons ◽

Reference Genomes

AbstractThe genetic basis of virulence in Mycobacterium tuberculosis has been investigated through genome comparisons of its virulent (H37Rv) and attenuated (H37Ra) sister strains. Such analysis, however, relies heavily on the accuracy of the sequences. While the H37Rv reference genome has had several corrections to date, that of H37Ra is unmodified since its original publication. Here, we report the assembly and finishing of the H37Ra genome from single-molecule, real-time (SMRT) sequencing. Our assembly reveals that the number of H37Ra-specific variants is less than half of what the Sanger-based H37Ra reference sequence indicates, undermining and, in some cases, invalidating the conclusions of several studies. PE_PPE family genes, which are intractable to commonly-used sequencing platforms because of their repetitive and GC-rich nature, are overrepresented in the set of genes in which all reported H37Ra-specific variants are contradicted. We discuss how our results change the picture of virulence attenuation and the power of SMRT sequencing for producing high-quality reference genomes.

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Ion channel profiling of the Lymnaea stagnalis ganglia via transcriptome analysis

10.21203/rs.3.rs-31358/v1 ◽

2020 ◽

Author(s):

Nan Dong ◽

Julia Bandura ◽

Zhaolei Zhang ◽

Yan Wang ◽

Karine Labadie ◽

...

Keyword(s):

Ion Channels ◽

Reference Genome ◽

Lymnaea Stagnalis ◽

De Novo ◽

Model Organisms ◽

Sequence Length ◽

Pond Snail ◽

Sequence Information ◽

Functional Domain ◽

Rna Seq

Abstract Background. The pond snail Lymnaea stagnalis (L. stagnalis) has been widely used as a model organism in neurobiology, ecotoxicology, and parasitology due to the relative simplicity of its CNS. However, its usefulness is restricted by a limited availability of transcriptome data. While sequence information for the L. stagnalis CNS transcripts has been obtained from EST library and a de novo RNA-seq assembly, the quality of these assemblies is limited by a combination of low coverage of EST libraries, the fragmented nature of de novo assemblies, and lack of reference genome. Results. In this study, taking advantage of the recent availability of the L. stagnalis reference genome, we generated an RNA-seq library from the adult L. stagnalis CNS, using a combination of genome-guided and de novo assembly programs to identify 17,832 protein-coding L. stagnalis transcripts. We combined our library with existing resources to produce a transcript set with greater sequence length, completeness, and diversity than previously available ones. Using our assembly and functional domain analysis, we profiled L. stagnalis CNS transcripts encoding ion channels and ionotropic receptors, which are key proteins for CNS function, and compared their sequences to other vertebrate and invertebrate model organisms. Interestingly, L. stagnalis transcripts encoding numerous putative Ca2+ channels showed the most sequence similarity to those of mouse, zebrafish, Xenopus tropicalis, fruit fly, and C. elegans, suggesting that many calcium channel-related signaling pathways may be evolutionarily conserved. Conclusions. Our study provides the most thorough characterization to date of the L. stagnalis transcriptome and provides insights into differences between vertebrates and invertebrates in CNS transcript diversity, according to function and protein class. Furthermore, this study is, to the best of our knowledge, the first to provide a complete characterization of the ion channels of a single species, opening new avenues for future research on fundamental neurobiological processes.

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