Generation of whole genome sequences of new Cryptosporidium hominis and Cryptosporidium parvum isolates directly from stool samples

BMC Genomics
Stephen J HadfieldRachel M Chalmers

Abstract

Whole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction, with subsequent use of the transposome-based Nextera XT kit to generate libraries for sequencing on Illumina platforms. IMS was found to be superior to caesium chloride density centrifugation for purification of oocysts from small volume stool samples and for reducing levels of contaminant DNA. The IMS-based method was used initially to sequence whole genomes of Cryptosporidium hominis gp60 subtype IbA10G2 and Cryptosporidium parvum gp60 subtype IIaA19G1R2 from small amounts of stool left over from diagnostic testing of clinical cases of cryptosporidiosis. The C. parvum isolate was sequenced to a mean depth of 51.8X with reads covering 100 % of the bases o...Continue Reading

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Citations

Sep 30, 2016·Euro Surveillance : Bulletin Européen Sur Les Maladies Transmissibles = European Communicable Disease Bulletin·R M Chalmers, S Cacciò
Jul 9, 2016·PLoS Neglected Tropical Diseases·Catrin E MooreRachel M Chalmers
Aug 31, 2016·Parasitology·Damer P Blake, Martha Betson
Nov 9, 2017·Parasitology·Jan Šlapeta
Mar 8, 2018·The Journal of Infectious Diseases·Carol A GilchristWilliam A Petri
Dec 23, 2016·Journal of Clinical Microbiology·Per SikoraRomanico B G Arrighi
Sep 13, 2019·PLoS Pathogens·Rodrigo P Baptista, Jessica C Kissinger
Mar 14, 2020·Parasite : Journal De La Société Française De Parasitologie·Giovanni WidmerLoïc Favennec
May 15, 2020·Journal of Clinical Microbiology·Higor Sette PereiraTiago Antônio de Oliveira Mendes
May 2, 2020·Scientific Reports·Christophe AudebertMagali Chabé
Nov 7, 2017·Frontiers in Microbiology·Camilla SekseJianxin Shi
Jan 11, 2020·Frontiers in Public Health·Arthur MorrisRachel M Chalmers
Oct 31, 2020·Microorganisms·Manasi SawantSadia Benamrouz-Vanneste
Jul 28, 2019·Trends in Parasitology·Sonja RueckertAnastasios D Tsaousis
Dec 24, 2020·Microbial Genomics·Swapnil TichkuleSimone Mario Caccio
Aug 22, 2021·The Journal of Infectious Diseases·Ines DettwilerPhilipp Olias

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Datasets Mentioned

BETA
PRJNA15585
PRJNA32283
PRJNA215218
PRJNA253845-PRJNA253848
KP280061

Methods Mentioned

BETA
density gradient centrifugation
PCR
Illumina sequencing
genotyping
Assay

Software Mentioned

PAGIT
BLAST
ABACAS
MS Excel
RATT
BLASTN
Basic Local Alignment Search Tool ( BLAST )
CLC Genomics Workbench
Bowtie2
IMAGE

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