Improved contiguity of the threespine stickleback genome using long-read sequencing

Nath, Shivangi; Shaw, Daniel E; White, Michael A.

doi:10.1101/2020.06.30.170787

Cited by 13 publications

(18 citation statements)

References 54 publications

(61 reference statements)

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“…However, it is also possible that the fusions were ancestral and that the greater number of chromosomes in some species results from chromosomal fission. To distinguish between these possibilities, we used the newly available whole-genome assemblies of the outgroup A. flavidus (Li et al submitted), P. pungitius (Varadharajan et al 2019), and G. aculeatus (Nath et al 2021), as well as the high-quality assembly of A. quadracus generated in this study. We then focused on the wholechromosome rearrangements that have occurred in G. aculeatus to determine whether these rearrangements are associated with genetic loci that underlie adaptation to divergent marine and freshwater habitats in this species.…”

Section: Phylogenetic Relationship and Chromosome Numbers Of Stickleback Speciesmentioning

confidence: 99%

“…For this study, we also used the available genome assemblies for G. aculeatus (Nath et al 2021)), P. pungitius (Varadharajan et al 2019), and the outgroup A. flavidus (Li et al submitted). We also used available RNA-seq data from G. nipponicus (Ishikawa et al 2019).…”

Section: Dna and Rna Extraction And Sequencingmentioning

confidence: 99%

“…The QTL data were filtered to remove redundant QTL following Rennison and Peichel (in review), and only the 655 QTL found in crosses between marine and freshwater populations were retained for the downstream analysis (Supplementary Table S2). We first mapped all the retained QTL with confidence intervals to the G. aculeatus v.5 genome (Nath et al 2021) in 50kb windows, following Peichel and Marques (2017). Next, we used the data from the original QTL papers to determine whether the marine allele at these QTL confers a marine phenotype and vice versa, which would suggest that these QTL contribute to adaptation to the divergent marine and freshwater habitats.…”

Section: Genomic Distribution Of Marine-freshwater Qtl In G Aculeatusmentioning

confidence: 99%

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Chromosomal Fusions Facilitate Adaptation to Divergent Environments in Threespine Stickleback

Liu

Roesti

Marques

et al. 2021

Molecular Biology and Evolution

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Chromosomal fusions are hypothesized to facilitate adaptation to divergent environments, both by bringing together previously unlinked adaptive alleles and by creating regions of low recombination that facilitate the linkage of adaptive alleles. But, there is little empirical evidence to support this hypothesis. Here, we address this knowledge gap by studying threespine stickleback (Gasterosteus aculeatus), in which ancestral marine fish have repeatedly adapted to freshwater across the northern hemisphere. By comparing the threespine and ninespine stickleback (Pungitius pungitius) genomes to a de novo assembly of the fourspine stickleback (Apeltes quadracus) and an outgroup species, we find two chromosomal fusion events involving the same chromosomes have occurred independently in the threespine and ninespine stickleback lineages. On the fused chromosomes in threespine stickleback, we find an enrichment of quantitative trait loci (QTL) underlying traits that contribute to marine versus freshwater adaptation. By comparing whole genome sequences of freshwater and marine threespine stickleback populations, we also find an enrichment of regions under divergent selection on these two fused chromosomes. There is elevated genetic diversity within regions under selection in the freshwater population, consistent with a simulation study showing that gene flow can increase diversity in genomic regions associated with local adaptation and our demographic models showing gene flow between the marine and freshwater populations. Integrating our results with previous studies, we propose that these fusions created regions of low recombination that enabled the formation of adaptative clusters, thereby facilitating freshwater adaptation in the face of recurrent gene flow between marine and freshwater threespine sticklebacks.

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Section: Phylogenetic Relationship and Chromosome Numbers Of Stickleback Speciesmentioning

confidence: 99%

Section: Dna and Rna Extraction And Sequencingmentioning

confidence: 99%

Section: Genomic Distribution Of Marine-freshwater Qtl In G Aculeatusmentioning

confidence: 99%

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Chromosomal Fusions Facilitate Adaptation to Divergent Environments in Threespine Stickleback

Liu

Roesti

Marques

et al. 2021

Molecular Biology and Evolution

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“…Illumina base call files for all libraries were converted to FASTQs using bcl2fastq v2.20.0.422 (Illumina) and FASTQ files were aligned to reference genome constructed from the v5 G. aculeatus assembly and annotation files available at https://stickleback.genetics.uga.edu/ (65). Briefly, annotations from Ensembl (release 95) were combined with repeat, Y chromosome, and revised annotations from Nath et al using AGAT (0.4.0) (66), and a STAR-compatible reference genome was generated Cell Ranger (v3.1.0, 10x Genomics) using these annotations and the v5 assembly from Nath et al The Cell Ranger count (v3.1.0) pipeline was used to construct cell-by-gene counts matrix for each library, subsequently analyzed using Scanpy 1.3.7 (67) and the Loupe Cell Browser (10x Genomics).…”

Section: Methodsmentioning

confidence: 99%

Single-cell RNA sequencing reveals micro-evolution of the stickleback immune system

Fuess

Bolnick

2021

Preprint

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Pathogenic infection is an important driver of many ecological processes. Furthermore, variability in immune function is an important driver of differential infection outcomes. New evidence would suggest that immune variation extends to broad cellular structure of immune systems. However, variability at such broad levels is traditionally difficult to detect in non-model systems. Here we leverage single cell transcriptomic approaches to document signatures of microevolution of immune system structure in a natural system, the three-spined stickleback (Gasterosteus aculeatus). We sampled nine adult fish from three populations with variability in resistance to a cestode parasite, Schistocephalus solidus, to create the first comprehensive immune cell atlas for G. aculeatus. Eight major immune cell types, corresponding to major vertebrate immune cells, were identified. We were also able to document significant variation in both abundance and expression profiles of the individual immune cell types, among the three populations of fish. This variability may contribute to observed variability in parasite susceptibility. Finally, we demonstrate that identified cell type markers can be used to reinterpret traditional transcriptomic data. Combined our study demonstrates the power of single cell sequencing to not only document evolutionary phenomena (i.e. microevolution of immune cells), but also increase the power of traditional transcriptomic datasets.

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“…Residual adapter sequences from the Nextera primers were trimmed using trimmomatic (v0.36) with PE and ILLUMINACLIP (keeping both reads) and the following parameters: LEADING:0 TRAILING:0 MINLEN:30 (Bolger et al 2014). Trimmed reads were aligned to the revised threespine stickleback genome (Nath et al 2020), including the mitochondrion and unplaced scaffolds, using Bowtie2 (v2.3.5; -X 1000 –no-unal) (Langmead and Salzberg 2012). Reads with a mapping quality less than 20 were filtered from the alignments using SAMtools (v1.10) (Li et al 2009).…”

Section: Methodsmentioning

confidence: 99%

Long-read RNA sequencing reveals widespread sex-specific alternative splicing in threespine stickleback fish

Shanfelter¹,

Pau

White

2020

Preprint

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Alternate isoforms contribute immensely to phenotypic diversity across eukaryotes. While short read RNA-sequencing has increased our understanding of isoform diversity, it is challenging to accurately detect full-length transcripts, preventing the identification of many alternate isoforms. Long-read sequencing technologies have made it possible to sequence full length alternative transcripts, accurately characterizing alternative splicing events, alternate transcription start and end sites, and differences in UTR regions. Here, we utilize PacBio long read RNA-sequencing (Iso-Seq) to examine the transcriptomes of five tissues in threespine stickleback fish (Gasterosteus aculeatus), a widely used genetic model species. The threespine stickleback fish has a refined genome assembly with gene annotations that are based on short-read RNA sequencing and predictions from coding sequence of other species. This suggests some of the existing annotations may be inaccurate or alternative transcripts may not be fully characterized. Using Iso-Seq we detected thousands of novel isoforms, indicating many isoforms are absent in the current Ensembl gene annotations. In addition, we refined many of the existing annotations within the genome. We noted many improperly positioned transcription start sites that were refined with long-read sequencing. The Iso-Seq predicted transcription start sites were more accurate, verified through ATAC-seq. We were also able to detect many alternative splicing events between sexes and across tissues. We found a substantial number of genes in both somatic and gonad tissue that had sex-specific isoforms. Our study highlights the power of long-read sequencing to study the complexity of transcriptomes, greatly improving genomic resources for the threespine stickleback fish.

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Improved contiguity of the threespine stickleback genome using long-read sequencing

Cited by 13 publications

References 54 publications

Chromosomal Fusions Facilitate Adaptation to Divergent Environments in Threespine Stickleback

Chromosomal Fusions Facilitate Adaptation to Divergent Environments in Threespine Stickleback

Single-cell RNA sequencing reveals micro-evolution of the stickleback immune system

Long-read RNA sequencing reveals widespread sex-specific alternative splicing in threespine stickleback fish

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