Novel workflow for metagenomics and transcriptomics analysis of A.D. systems

Sammanfattning: The A.D. systems (anaerobic digestion), when used in biogas reactors, are an advanced ecological way to produce energy while treating waste. The majority of the microbial community of the reactor remains unknown to this day, due to the impossibility to culture most of the bacteria individually. Metagenomics and transcriptomics aim to discover those bacteria and understand the interactions within the community. HTS (high throughput sequencing) technology opens new possibilities in terms of length of the reads sequenced and accuracy. Sequencing done by Oxford Nanopore machines can produce long reads while having a slightly worse accuracy than other machines, where Illumina sequencing machines have a higher accuracy to the detriment of lengths. The two sequencing methods complement each other, and the hybrid assembly uses both long and short reads to create longer and more accurate contigs that can then be further analysed. Here is presented a metagenomics pipeline (MUFFIN) based on the hybrid assembly of short and long reads followed by multiple differential binning methods and refinement to produce high-quality bins and their annotations. The pipeline is written by using Nextflow to achieve high reproducibility and fast and straightforward use of the pipeline. This pipeline also produces the taxonomic classification of the bins as well as a transcription, quantification and annotation of RNAseq data. The pipeline was tested using one biogas reactor as an example to assess the capacity of MUFFIN to process and output relevant files needed to analyse the microbial community and their function. A parsing script was developed to analyse and summarise the annotations files. The script outputs a quantification file of the transcripts annotated, an HTML file summarising the pathways across the bins and transcripts, and an HTML file for each bin summarising the annotation.