Hi all! I'm a biology student doing a bird gut microbiome sequence analysis using 16S v4 region (amplified by 515F-806R primers, Illumina MiSeq). My basic objective is to prolife the bird gut microbiome in time series and perform functional analysis prediction using PICRUSt2. Due to limitations of my laptop's computational power, I'm using Nephele (Nephele) to analyze my data via QIIME2 as this is the only platform I found that I can use QIIME2 and PICRUSt2 (and for free of charge). I tried localizing QIIME2 galaxy but it seems my 8GB RAM laptop couldn't really make it run.
So my situation: I noticed in my tax bar plot that very small percentage of two of my samples are archaeal sequences and Nephele has no parameters in it's UI that I can modify to filter them out. I've been reading about QIIME2 filtering reads command and in most QIIME2 tutorials for 16S metabarcording, filtering reads were only being done for Mitochondrial and Chloroplast sequences (as opposed to my experience in mothur in Galaxy where Archaea, Eukaryotes, Mitochondrial, and Chloroplasts were being filtered out).
I know that in reality, archaea microbes are part of gut microbiota but two of my bird gut samples (collected in the same timepoint, 2 out of 5 samples in the same time point) have very very small portion of archaeal sequences (like only 10 featured IDs of archaeal sequences from taxonomy.qzv out of 4,525) that it looks like an artefact rather than true result. My readings on 515F-806R primers seem that they can amplify archaeal sequences, though. So I'm really torn if this is worth moving forward. This is my first time doing this analyses and pretty much doing fine til I met with the Archeal sequences dilemma since I don't know how to move forward with the analysis knowing these sequences exists in my samples. I have no technical background on bioinformatics (though I've been really trying hard to understand it). I don't have access to Ubuntu and running VirtualBox is definitely out of the options due to memory and storage issues. Also, I will light up my laptop to fire as it heats up pretty much when I tried doing the Galaxy docker for QIIME2 which I pretty much left it overnight running but it didn't progress much. I also reached out to Nephele still awaiting response.
Any inputs and tips will be greatly appreciated! Thanks and Regards!
Here's the parameters I've indicated in the run:
|o|data_type : QIIME2_16S_PE |
|o|min_frequency : 2000 |
|o|min_quality : 20 |
|o|otu_reference : 97 |
|o|otu_strategy : closed |
|o|perc_identity : 0.97 |
|o|phylogenetic : sepp |
|o|sepp_reference : silva |
|o|sklearn_options : silva_full |
|o|taxonomy_methods : sklearn |
Another batch of my samples also contains archaea, this time 23 Featured IDs out of over 7000 reads are archaeans in three samples.
I really don't know if archaeans are naturally occurring in 16S V4 metabarcoding so I'm kind of bothered by this, particularly I don't have the means to perform filtering step unless I can try attempting doing QIIME2 docker which really don't turn out well with my previous attempts installing it.