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ARS Home » Midwest Area » Madison, Wisconsin » U.S. Dairy Forage Research Center » Cell Wall Biology and Utilization Research » Research » Publications at this Location » Publication #412902
Research Project: Developing Strategies to Improve Dairy Cow Performance and Nutrient Use Efficiency with Nutrition, Genetics, and Microbiology

Location: Cell Wall Biology and Utilization Research

Title: In search of a better way: Exploring the impact of enrichment and DNA extraction on long-read sequencing of rumen protozoa.

Author
item McClure, Jennifer
item Panke-Buisse, Kevin
item LAL, ELLEN - Abs Global
item AMORIN DE HEGEDUS, ROCIO - Oak Ridge Institute For Science And Education (ORISE)
item Zanton, Geoffrey
item French, Elizabeth
item Smith, Timothy - Tim
item SUEN, GARRET - University Of Wisconsin
item BICKHART, DEREK - Hendrix Genetics

Submitted to: American Dairy Science Association Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 3/18/2024
Publication Date: 6/16/2024
Citation: McClure, J., Panke-Buisse, K. Lai, E., deHegedus, R.A., Zanton, G., French, E., Smith, T., Suen, G., Bickhart, D. 2024. In search of a better way: Exploring the effects of enrichment and DNA extraction on long-read sequencing of rumen protozoa [abstract]. Journal of Dairy Science. 107 (Supplement 1):268

Interpretive Summary:

Technical Abstract: Protozoa constitute up to 50% of the living biomass in the rumen, though the roles they play are largely unknown. While advances in long-read sequencing and metagenomics have provided resources for rumen bacteria, fungi and archaea, protozoa continue to present unique challenges. Their genomes’ high AT-richness and repeat regions result in biases during DNA extraction, sequencing and downstream analysis. Enrichment of protozoa from rumen liquid (RL) can mitigate biases from extraction and sequencing by increasing the proportion and quality of extracted DNA. A gravity-based separation and low-speed centrifugation method optimized for downstream long-read sequencing was developed to enrich protozoa in RL samples. DNA from enriched and unenriched samples from 2 multiparous Holstein cows were extracted via seven distinct protocols (kits from Invitrogen (INV), Millipore (MIL), Promega (PRO), Qiagen (QIA), Roche (ROC), ZYMO (ZYM), and a modified sodium dodecyl sulfate lysis (YM)) to identify suitability for downstream Oxford Nanopore Technologies (ONT) sequencing and potential biases. Extracted DNA samples were sequenced on ONT R9.4.1 flow cells, reads were classified and annotated with Kaiju, and results were visualized in Krona. Fragment length distribution of extracted DNA was sufficient for downstream ONT sequencing for six extraction protocols. For the six protocols, enrichment resulted in an average increase in AT-richness of 13.8+/-0.17%. The proportion of classified Eukaryotic reads in enriched samples were five-fold greater than unenriched samples (~32% and ~6%, respectively). There were statistically significant differences between sequenced fragment length for all kits (p<0.05). YM and ROC produced the longest reads on average, 4889 bp and 4275 bp respectively, though ROC was more consistent between animals. The proportions of sequenced reads that could be classified were relatively small and highlight the need for additional information on rumen protozoa to improve classification databases. However, these results indicate enrichment and ONT long-read sequencing are viable methods for obtaining high-quality protozoa metagenomic data from RL.