I've been given occasion to plunge into the metagenomics field. I'm working with shotgun sequencing data, so not targeted at 16S or another gene. From reading several articles, including this review by Siegwald et al., and the websites of QIIME and mothur, it seems that targeted (non-shotgun) analyses are the standard, but that I cannot use the same software to analyse shotgun sequencing data.
Q1: Is this correct?
To analyze shotgun sequencing, I've so far identified two tools: Kraken2 and MetaPhlAn2. I know that there are other tools available such as CLARK, but they do not seem to be significantly different from Kraken2.
After trying Kraken2, it only seems to support export to the Krona visualization tool for downstream analysis. So I now intend to try MetaPhlAn2. Output from this tool should be compatible with the R packages phyloseq and microbiome, which seem to be standard tools for downstream statistical analyses of metagenomes.
Q2: Are the observations and imputations in this paragraph correct? Q3: Should I indeed be aiming for compatibility with phyloseq and microbiome, or are there alternatives for downstream statistical analysis? Q4: Is it indeed not possible to do further statistical analysis on Kraken2's output?
Finally, the datasets which I'm starting my analyses from are not so large, so the speed advantage of Kraken2 compared to other tools does not matter much to me. I'm mostly interested in increasing the sensitivity of my analysis. Human reads have already been removed from the datasets (BAMs) by alignment, although Kraken2 could still identify a significant share.
Thank you for your time!
P.S. I've read this recent review by Ye et al., but it mostly discusses taxonomic classifiers performance, not the possibilities for downstream statistical analysis.