Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F. tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing

Abstract BackgroundThe identification of environmentally specific alleles and the observation of evolutional processes is a goal of conservation genomics. By generational changes of allele frequencies in populations, questions regarding effective population size, gene flow, drift, and selection can be addressed. The observation of such effects often is a trade-off of costs and resolution, when a decent sample of genotypes should be genotyped for many loci. Pool genotyping approaches can derive a high resolution and precision in allele frequency estimation, when high coverage sequencing is utilized. Still, pool high coverage pool sequencing of big genomes comes along with high costs.ResultsHere we present a reliable method to estimate a barley population’s allele frequency at low coverage sequencing. Three hundred genotypes were sampled from a barley backcross population to estimate the entire population’s allele frequency. The allele frequency estimation accuracy and yield were compared for three next generation sequencing methods. To reveal accurate allele frequency estimates on a low coverage sequencing level, a haplotyping approach was performed. Low coverage allele frequency of positional connected single polymorphisms were aggregated to a single haplotype allele frequency, resulting in two to 271 times higher depth and increased precision. We compared different haplotyping tactics, showing that gene and chip marker-based haplotypes perform on par or better than simple contig haplotype windows. The comparison of multiple pool samples and the referencing against an individual sequencing approach revealed whole genome pool resequencing having the highest correlation to individual genotyping (up to 0.97), while transcriptomics and genotyping by sequencing indicated higher error rates and lower correlations.ConclusionUsing the proposed method allows to identify the allele frequency of populations with high accuracy at low cost. This is particularly interesting for conservation genomics in species with big genomes, like barley or wheat. Whole genome low coverage resequencing at 10x coverage can deliver a highly accurate estimation of the allele frequency, when a loci-based haplotyping approach is applied. Using annotated haplotypes allows to capitalize from biological background and statistical robustness.

Download Full-text

Erratum to: Whole Genome DNA Methylation Analysis of Brachypodium distachyon Using Next-Generation Sequencing (BS-seq)

Brachypodium Genomics - Methods in Molecular Biology ◽

10.1007/978-1-4939-7278-4_19 ◽

2018 ◽

pp. E1-E1

Author(s):

I-Hsuan Lin

Keyword(s):

Dna Methylation ◽

Next Generation Sequencing ◽

Brachypodium Distachyon ◽

Whole Genome ◽

Next Generation ◽

Methylation Analysis ◽

Dna Methylation Analysis ◽

Generation Sequencing

Download Full-text

Genome-wide characterization of copy number variations in the host genome in genetic resistance to Marek's disease using next generation sequencing

10.21203/rs.2.12741/v2 ◽

2020 ◽

Author(s):

Hao Bai ◽

Yanghua He ◽

Yi Ding ◽

Huanmin Zhang ◽

Jilan Chen ◽

...

Keyword(s):

Next Generation Sequencing ◽

Copy Number ◽

Marek's Disease ◽

Neoplastic Disease ◽

Marek’S Disease ◽

Next Generation ◽

Qrt Pcr ◽

Genome Wide ◽

A Genome ◽

Generation Sequencing

Abstract Background: Marek’s disease (MD) is a highly neoplastic disease primarily affecting chickens, and remains as a chronic infectious disease that threatens the poultry industry. Copy number variation (CNV) has been examined in many species and is recognized as a major source of genetic variation that directly contributes to phenotypic variation such as resistance to infectious diseases. Two highly inbred chicken lines 63 (MD-resistant) and 72 (MD-susceptible), as well as their F1 generation and six recombinant congenic strains (RCSs) with varied susceptibility to MD, are considered as ideal models to identify the complex mechanisms of genetic and molecular resistance to MD.Results: In the present study, to unravel the potential genetic mechanisms underlying resistance to MD, we performed a genome-wide CNV detection using next generation sequencing on the inbred chicken lines with the assistance of CNVnator. As a result, a total of 1,649 CNV regions (CNVRs) were successfully identified after merging all the nine datasets, of which 90 CNVRs were overlapped across all the chicken lines. Within these shared regions, 1,360 harbored genes were identified. In addition, 55 and 44 CNVRs with 62 and 57 harbored genes were specifically identified in line 63 and 72, respectively. Bioinformatics analysis showed that the nearby genes were significantly enriched in 36 GO terms and 6 KEGG pathways including JAK/STAT signaling pathway. Ten CNVRs (nine deletions and one duplication) involved in 10 disease-related genes were selected for validation by using qRT-PCR, all of which were successfully confirmed. Finally, qRT-PCR was also used to validate two deletion events in line 72 that were definitely normal in line 63. One high-confidence gene, IRF2 was identified as the most promising candidate gene underlying resistance and susceptibility to MD in view of its function and overlaps with data from previous study.Conclusions: Our findings provide valuable insights for understanding the genetic mechanism of resistance to MD and the identified gene and pathway could be considered as the subject of further functional characterization.

Download Full-text