Long-read assembly and comparative evidence-based reanalysis of Cryptosporidium genome sequences reveal new biological insights

ABSTRACTCryptosporidiosis is a leading cause of waterborne diarrheal disease globally and an important contributor to mortality in infants and the immunosuppressed. Despite its importance, the Cryptosporidium community still relies on a fragmented reference genome sequence from 2004. Incomplete reference sequences hamper experimental design and interpretation. We have generated a new C. parvum IOWA genome assembly supported by PacBio and Oxford Nanopore long-read technologies and a new comparative and consistent genome annotation for three closely related species C. parvum, C. hominis and C. tyzzeri. The new C. parvum IOWA reference genome assembly is larger, gap free and lacks ambiguous bases. This chromosomal assembly recovers 13 of 16 possible telomeres and raises a new hypothesis for the remaining telomeres and associated subtelomeric regions. Comparative annotation revealed that most “missing” orthologs are found suggesting that species differences result primarily from structural rearrangements, gene copy number variation and SNVs in C. parvum, C. hominis and C. tyzzeri. We made >1,500 C. parvum annotation updates based on experimental evidence. They included new transporters, ncRNAs, introns and altered gene structures. The new assembly and annotation revealed a complete DNA methylase Dnmt2 ortholog. 190 genes under positive selection including many new candidates were identified using the new assembly and annotation as reference. Finally, possible subtelomeric amplification and variation events in C. parvum are detected that reveal a new level of genome plasticity that will both inform and impact future research.

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Long-read assembly and comparative evidence-based reanalysis of Cryptosporidium genome sequences reveals expanded transporter repertoire and duplication of entire chromosome ends including subtelomeric regions

Genome Research ◽

10.1101/gr.275325.121 ◽

2021 ◽

pp. gr.275325.121

Author(s):

Rodrigo P. Baptista ◽

Yiran Li ◽

Adam Sateriale ◽

Karen L. Brooks ◽

Alan Tracey ◽

...

Keyword(s):

Genome Assembly ◽

Reference Genome ◽

Diarrheal Disease ◽

Gene Copy ◽

Future Research ◽

Single Nucleotide Variants ◽

Cryptosporidium Hominis ◽

Entire Chromosome ◽

Long Read ◽

Subtelomeric Regions

Cryptosporidiosis is a leading cause of waterborne diarrheal disease globally and an important contributor to mortality in infants and the immunosuppressed. Despite its importance, the Cryptosporidium community has only had access to a good, but incomplete, Cryptosporidium parvum IOWA reference genome sequence. Incomplete reference sequences hamper annotation, experimental design and interpretation. We have generated a new C. parvum IOWA genome assembly supported by PacBio and Oxford Nanopore long-read technologies and a new comparative and consistent genome annotation for three closely related species C. parvum, Cryptosporidium hominis and Cryptosporidium tyzzeri. We made 1,926 C. parvum annotation updates based on experimental evidence. They include new transporters, ncRNAs, introns and altered gene structures. The new assembly and annotation revealed a complete Dnmt2 methylase ortholog. Comparative annotation between C. parvum, C. hominis and C. tyzzeri revealed that most "missing" orthologs are found suggesting that the biological differences between the species must result from gene copy number variation, differences in gene regulation and single nucleotide variants (SNVs). Using the new assembly and annotation as reference, 190 genes are identified as evolving under positive selection, including many not detected previously. The new C. parvum IOWA reference genome assembly is larger, gap free and lacks ambiguous bases. This chromosomal assembly recovers all 16 chromosome ends, 13 of which are contiguously assembled. The three remaining chromosome ends are provisionally placed. These ends represent duplication of entire chromosome ends including subtelomeric regions revealing a new level of genome plasticity that will both inform and impact future research.

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Long read sequencing reveals Poxvirus evolution through rapid homogenization of gene arrays

10.1101/245373 ◽

2018 ◽

Author(s):

Thomas A. Sasani ◽

Kelsey R. Cone ◽

Aaron R. Quinlan ◽

Nels C. Elde

Keyword(s):

Copy Number ◽

Gene Copy Number ◽

Gene Copy ◽

Single Nucleotide Variants ◽

Sequencing Platform ◽

Sequencing Technologies ◽

Gene Copy Number Variation ◽

Large Gene ◽

Long Read ◽

Number Variation

AbstractLarge DNA viruses rapidly evolve to defeat host defenses. Poxvirus adaptation can involve combinations of recombination-driven gene copy number variation and beneficial single nucleotide variants (SNVs) at the same locus, yet how these distinct mechanisms of genetic diversification might simultaneously facilitate adaptation to immune blocks is unknown. We performed experimental evolution with a vaccinia virus population harboring a SNV in a gene actively undergoing copy number amplification. Comparisons of virus genomes using the Oxford Nanopore Technologies sequencing platform allowed us to phase SNVs within large gene copy arrays for the first time, and uncovered a mechanism of adaptive SNV homogenization reminiscent of gene conversion, which is actively driven by selection. Our work reveals a new mechanism for the fluid gain of beneficial mutations in genetic regions undergoing active recombination in viruses, and illustrates the value of long read sequencing technologies for investigating complex genome dynamics in diverse biological systems.

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Long read sequencing reveals poxvirus evolution through rapid homogenization of gene arrays

eLife ◽

10.7554/elife.35453 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 5

Author(s):

Thomas A Sasani ◽

Kelsey R Cone ◽

Aaron R Quinlan ◽

Nels C Elde

Keyword(s):

Copy Number ◽

Gene Copy Number ◽

Gene Copy ◽

Single Nucleotide Variants ◽

Sequencing Technologies ◽

Gene Copy Number Variation ◽

Large Gene ◽

Oxford Nanopore ◽

Long Read ◽

Number Variation

Poxvirus adaptation can involve combinations of recombination-driven gene copy number variation and beneficial single nucleotide variants (SNVs) at the same loci. How these distinct mechanisms of genetic diversification might simultaneously facilitate adaptation to host immune defenses is unknown. We performed experimental evolution with vaccinia virus populations harboring a SNV in a gene actively undergoing copy number amplification. Using long sequencing reads from the Oxford Nanopore Technologies platform, we phased SNVs within large gene copy arrays for the first time. Our analysis uncovered a mechanism of adaptive SNV homogenization reminiscent of gene conversion, which is actively driven by selection. This study reveals a new mechanism for the fluid gain of beneficial mutations in genetic regions undergoing active recombination in viruses and illustrates the value of long read sequencing technologies for investigating complex genome dynamics in diverse biological systems.

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Faculty Opinions recommendation of Diet and the evolution of human amylase gene copy number variation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1092307.546722 ◽

2007 ◽

Author(s):

Magnus Ingelman-Sundberg

Keyword(s):

Copy Number Variation ◽

Copy Number ◽

Gene Copy Number ◽

Gene Copy ◽

Amylase Gene ◽

Gene Copy Number Variation ◽

Number Variation

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What a finding of gene copy number variation can add to the diagnosis of developmental neuropsychiatric disorders

Current Opinion in Genetics & Development ◽

10.1016/j.gde.2020.12.017 ◽

2021 ◽

Vol 68 ◽

pp. 18-25

Author(s):

Jacob Vorstman ◽

Stephen W Scherer

Keyword(s):

Copy Number Variation ◽

Copy Number ◽

Gene Copy Number ◽

Neuropsychiatric Disorders ◽

Gene Copy ◽

Gene Copy Number Variation ◽

Number Variation

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Induction of direct repeat recombination by psoralen–DNA adducts in Saccharomyces cerevisiae: Defects in DNA repair increase gene copy number variation

DNA Repair ◽

10.1016/j.dnarep.2014.05.011 ◽

2014 ◽

Vol 21 ◽

pp. 87-96 ◽

Cited By ~ 2

Author(s):

Wilma A. Saffran ◽

Anam Ahmed ◽

Olga Binyaminov ◽

Cynthia Gonzalez ◽

Amita Gupta ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Repair ◽

Copy Number Variation ◽

Dna Adducts ◽

Copy Number ◽

Gene Copy Number ◽

Direct Repeat ◽

Gene Copy ◽

Gene Copy Number Variation ◽

Number Variation

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Genetic variability of CYP2D6, CYP3A4 and CYP3A5 among the Egyptian population

Pharmacogenomics ◽

10.2217/pgs-2020-0140 ◽

2021 ◽

Author(s):

Thuraya M Mutawi ◽

Mohamed M Zedan ◽

Raida S Yahya ◽

Mahmoud M Zakria ◽

Mamdouh R El-Sawi ◽

...

Keyword(s):

Genetic Variability ◽

Copy Number Variation ◽

Copy Number ◽

Middle Eastern ◽

Gene Copy Number ◽

Gene Copy ◽

Allelic Variants ◽

Gene Copy Number Variation ◽

Number Variation ◽

Study Participants

Aim: This study investigated major allelic variants of CYP2D6, CYP3A4 and CYP3A5 in Egyptians, an Arabic population for which there is little information regarding these important pharmacogenes. Patients & methods: CYP2D6*2, *4, *5, *10, *41 and gene copy number variation, as well as CYP3A4*22 and CYP3A5*3 were determined with commercially available TaqMan assays in 145 healthy study participants. Results: The CYP2D6 alleles identified suggest that the prevalence of poor metabolizers is low as none were found among the 145 subjects investigated. The frequency for CYP3A5 nonexpressers was 74.5% and the CYP3A4*22 allele frequency was low at 2.0%. Conclusion: These preliminary findings indicate that pharmacogene variation in Egyptians is different from those of other Middle Eastern/Arabic populations and warrants further investigation.

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