Targeted enrichment outperforms other enrichment techniques and enables more multi-species RNA-Seq analyses

Abstract

Enrichment methodologies enable analysis of minor members in multi-species transcriptomic analyses. We compared standard enrichment of bacterial and eukaryotic mRNA to targeted enrichment with Agilent SureSelect (AgSS) capture for Brugia malayi, Aspergillus fumigatus, and the Wolbachia endosymbiont of B. malayi (wBm). Without introducing significant systematic bias, the AgSS quantitatively enriched samples, resulting in more reads mapping to the target organism. The AgSS-enriched libraries consistently had a positive linear correlation with its unenriched counterpart (r2=0.559-0.867). Up to a 2,242-fold enrichment of RNA from the target organism was obtained following a power law (r2=0.90), with the greatest fold enrichment achieved in samples with the largest ratio difference between the major and minor members. While using a single total library for prokaryote and eukaryote in a single sample could be beneficial for samples where RNA is limiting, we observed a decrease in reads mapping to protein coding genes and an increase of multi-mapping reads to rRNAs in AgSS enrichments from eukaryotic total RNA libraries as opposed to eukaryotic poly(A)-enriched libraries. Our results support a recommendation of using Agilent SureSelect targeted enrichment on poly(A)-enriched libraries for eukaryotic captures and total RNA libraries for prokaryotic captures to increase the robustness of multi-species transcriptomic studies.

Improved metagenome assemblies through selective enrichment of bacterial genomic DNA from eukaryotic host genomic DNA using ATAC-seq

“…The peaks are highly enriched in gene promoters, with a proportion of 46.2%. This enrichment is higher than what is typically observed in mammals (Chung et al, 2018;Yan et al, 2020). Similar to B. malayi, nearly 50% of peaks fall in promoter regions in Drosophila melanogaster (Santiago et al, 2021;Dhall et al, 2023).…”

“…An enrichment of peaks at promoter regions has been observed in other ATAC-seq datasets. In mammals, around 10-25% of peaks are found in promoter regions (Chung et al, 2018;Yan et al, 2020). Drosophila melanogaster data is more similar to B. malayi, where 40-50% of peaks are found in promoters (Santiago et al, 2021;Dhall et al, 2023).…”

A Meta-Analysis of Wolbachia Transcriptomics Reveals a Stage-Specific Wolbachia Transcriptional Response Shared Across Different Hosts

“…In the vector life stages of filarial nematodes, assessing the Wolbachia transcriptome is difficult due to the difference in abundance of both vector and nematode reads relative to Wolbachia reads (Choi et al 2014), such that rRNA-, poly(A)-depletions are inadequate in enriching for a sufficient number of Wolbachia reads. To address this, Agilent SureSelect RNA-Seq captures were designed for wBm, in order to capture the wBm transcriptional profile for samples with extremely low Wolbachia abundance (Chung et al 2018b). Using the Agilent SureSelect capture and rRNA-, poly(A)-depletions, the wBm transcriptome was able to be profiled across the entire B. malayi life cycle, consisting of 12 samples from the B. malayi mammalian life stages and three samples from the vector life stages.…”

“…As the first Wolbachia transcriptomics study was published in 2012 (Darby et al 2012), this discordance could be attributed to a difference in sequencing technologies and analysis tools over the past 8 years. Prior to the development of enrichment techniques, such as targeted RNA-Seq captures (Martin et al 2016;Chung et al 2018b), older Wolbachia transcriptomics studies may not have been able to sequence Wolbachia to a sufficient depth for differential expression analyses. Additionally, the more recent application of saturation curves to RNA-Seq analyses has proven to be a robust method to assess whether samples have been sequenced to a sufficient depth (Sims et al 2014) and dual-species transcriptomics studies benefit from the implementation of a rigorous minimum expression filter.…”

Long-read RNA sequencing of human and animal filarial parasites improves gene models and discovers operons

“…Short-read RNA sequencing data from across the B. malayi life cycle [46] were acquired from NCBI SRA; reads were remapped to Bmal-4.0 and counts were generated with HISAT2 [47] and…”