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Background <p><i>Cryptosporidium</i> is a protozoan parasite that causes diarrheal illness in humans and other animals and has a low infectious dose. The development of new tools to study and mitigate <i>Cryptosporidium</i> infection is aided by access to a diverse library of genomic material. However, culture methods to amplify the quantity of cells available from an individual isolate are not available, so <i>Cryptosporidium</i> DNA often falls below the input threshold for many genome sequencing methods.</p> Methods and results <p>Here, we assess the suitability of whole genome amplification (WGA) free methods of obtaining high quality whole genome sequences from low inputs of DNA from <i>Cryptosporidium</i>. Three commercially available library kits from Illumina, New England Biolabs (NEB), and Qiagen were compared. Testing was conducted on extractions from <i>Cryptosporidium</i> oocyst counts ranging from 1&#xa0;million (<i>n</i> = 1) to 1 oocyst (<i>n</i> = 4 for 100,000 and <i>n</i> = 8 for all other cell levels), representing DNA inputs ranging from approximately 5 ng to 0.005 pg. Similar performance was observed for the Illumina and NEB library prep kits for which 99% coverage of the reference genome was obtained from isolates containing between 1&#xa0;million and 1,000 oocysts. Because only 36% of the Qiagen preps produced successful libraries, sequencing was not performed on this group.</p> Conclusions <p>This research indicates that high quality <i>Cryptosporidium</i> whole genome sequences can be generated from DNA extracted from as few as 1,000 oocysts, representing an estimated 5 pg of input DNA, without the need for WGA, creating new avenues for sequencing genomes from samples with limited DNA content.</p>

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Whole genome sequencing of Cryptosporidium from low DNA inputs without whole genome amplification

  • 期刊论文

  • 发表日期：

    2025年12月29日

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  • Katherine Hshieh,
  • Aleksey Molokin,
  • Jenny G. Maloney

摘要

Background

Cryptosporidium is a protozoan parasite that causes diarrheal illness in humans and other animals and has a low infectious dose. The development of new tools to study and mitigate Cryptosporidium infection is aided by access to a diverse library of genomic material. However, culture methods to amplify the quantity of cells available from an individual isolate are not available, so Cryptosporidium DNA often falls below the input threshold for many genome sequencing methods.

Methods and results

Here, we assess the suitability of whole genome amplification (WGA) free methods of obtaining high quality whole genome sequences from low inputs of DNA from Cryptosporidium. Three commercially available library kits from Illumina, New England Biolabs (NEB), and Qiagen were compared. Testing was conducted on extractions from Cryptosporidium oocyst counts ranging from 1 million (n = 1) to 1 oocyst (n = 4 for 100,000 and n = 8 for all other cell levels), representing DNA inputs ranging from approximately 5 ng to 0.005 pg. Similar performance was observed for the Illumina and NEB library prep kits for which 99% coverage of the reference genome was obtained from isolates containing between 1 million and 1,000 oocysts. Because only 36% of the Qiagen preps produced successful libraries, sequencing was not performed on this group.

Conclusions

This research indicates that high quality Cryptosporidium whole genome sequences can be generated from DNA extracted from as few as 1,000 oocysts, representing an estimated 5 pg of input DNA, without the need for WGA, creating new avenues for sequencing genomes from samples with limited DNA content.