Separation Anxiety and Banding on Gels?

[Mehrbod_Estaki]

Hi @ChrisKeefe ,
Yikes!
The first thing that pops to my head, especially because this is separating so clearly on UU (and I'm guessing on Jaccard as well), is the host contamination. Are these Deblur or DADA2 denoised?
With Deblur, I wouldn't expect to see this so clearly because of its inbuilt positive filtering. With DADA2 if there are host contaminations it would obviously be included and so you'd have to remove them yourself. Have you looked at the taxonomies yet? BLAST a bunch of the unknown or unclassified sequences, in my experience, if you're using beads to disrupt the cells, some mouse DNA can get amplified and show up in your results like this. For gut samples I generally use a positive filter + do a taxonomy filter to remove anything that doesn't have at least Phylum level classification and that seems to do ok.

Some other thoughts:

@ChrisKeefe

Do any of you lovely people have experience with gels like these? Any insights into why this might be occurring, or how we might correct for it in future runs?

You can try doing a 2 step cell disruption to minimize host contamination. Step 1) cut tissue samples into smaller bits with scissors and do a high intense vortex (no beads) step with some detergent. This should ideally remove bacteria from the tissue sample and then you can separate the remaining big tissue chunks either by a short centrifuge, or try to pipette around it. Step 2) Your usual bead beating extraction without all the host cells there. The issue here is that this will require some benchmarking on your end to make sure you're not biasly discarding some bacterial cells alongside the host tissue. This may also have the unwanted consequence that will make it look different compared to previous data that simply "discarded the unknown".

@ChrisKeefe

Are there other likely explanatory variables we’ve overlooked here that we should explore?

Date of animal batches? Same vendor over time can still see change in microbiome (though certainly not THIS much)

Finally, can you clarify this gel by the way?Are the brightest bands the V4 target? What are the bands above and below it? What is their weight? Adding ladder weights to the image could help Also, are the wells in between the bright ones your no template controls? It's good that those are coming off blank, supports the idea of host contamination more.

@ChrisKeefe

Based on some QC figures we looked over, roughly 1/37 of the amplified sequences are between 600 and 700 bp long just before sequencing

This is strange indeed, though gels are not super accurate for that. You may want to look at some of them on a Bioanalzyer or something. What is your expected band weight with the target+barcodes+primers+adapters +linkers included?
Also, those gels can run weird on the outside wells, so you can try putting one of our ladders in a middle well, though I don't think this is the issue here. It could just be a not a perfect ladder?