Testing different methods for extracting symbiont reads from a *M. capitata* holobiont transcriptome

Alignment to a concatenated reference transcriptome compared to extraction from a de novo transcriptome assembly

Overview

Goal: To extract symbiont reads from a M. capitata holobiont transcriptome for differential gene expression analysis and subsequent functional enrichment analysis.

Methods: Here, I outline three pipelines that I used to extract symbiont reads from a M. capitata holobiont transcriptome, 1) alignment to a concatenated reference genome, 2) alignment to a concatenated reference transcriptome, and 3) extraction from a de novo transcriptome assembly. RNA was extracted using a Zymo-Duet Extraction Kit; see my notebook post, Protocol for DNA/RNA Extractions of Montipora Coral Larvae Using Zymo-Duet Extraction Kit, for the extraction protocol. The reads were then sent to Genewiz for library preparation (TruSeq Stranded mRNA Sample Preparation Protocol) and sequencing on a HiSeq instrument targeting 15 million reads per sample. Sequences were then quality assessed and cleaned using the pipeline outlined in M. capitata RNAseq QC, Alignment, Assembly Bioinformatic Pipeline.

Results: While I found that symbiont read coverage from the de novo assembly extraction exceded those from alignment to a concatenated reference transcriptome and genome, ultimately, coverage was too low for robust downstream analysis. However, coverage may be improved using other methods such as GC-content peak inference, as used by Frazier et al. (2017) or through other de novo assembly programs. Additionally, steps to enrich the symbiont reads during the RNA extraction process that were not taken here (e.g., through pelleting) could additionally lead to higher symbiont coverage. If working with coral tissues, this may be an extra measure you will want to take to ensure adequate symbiont RNA concentrations for differential expression analysis.

Necessary Programs and Equipment

  • A high performace computer (i.e. the URI bluewaves server; Instructions to obtain server access here) with the following programs:

Method 1: Extraction from concatenated reference genome:

Method 2: Extraction from concatenated reference transcriptome:

Method 3: Extraction from a de novo transcriptome:

References

Frazier, M., Helmkampf, M., Bellinger, M. R., Geib, S. M., & Takabayashi, M. (2017). De novo metatranscriptome assembly and coral gene expression profile of Montipora capitata with growth anomaly. BMC Genomics, 18(1). https://doi.org/10.1186/s12864-017-4090-y

:fa-Dolphin:

Written on July 2, 2021