First, a hearty thank you in advance for reading this and providing any assistance.

I've been tasked by my PI with sequencing a new bacterial isolate and verifying that it's a new species. I did the Illumina Nextera XT prep with the help of a labmate and got great reads on my first try. Awesome. I was stuck actually doing anything with the data for a long time until I found this incredibly helpful post and began sifting through the information in the comments. I have a greater appreciation for NGS and bioinformatics as a whole.

But I'm still stuck. I see lots of wonderful recommendations and debate on which tools are useful, which databases to query, Linux command line versus Windows GUI, etc. But I sit down with the programs I have available and spend 30 minutes trying to figure out why *.fas will import but *.fa won't. I had my first "sitting in lab on the verge of tears" moment yesterday (yay!), so I'm reaching out.

What I have:

• Two unpaired *.fastq sequences from Illumina, ostensibly aligned in CLC
• CLC versions 7-11 (why? we'll never know)
• Mega7

What I need:

• Is this bug a new species?

I know, my PI should be providing some support. Yes, it's odd that the lab has these tools and no one really knows how to use them. Yes, being told the answer is not the greatest way to learn a new thing. But please, tell me what buttons to press, or point me towards a workflow/recipe I can follow. I understand 16S is the standard for this question, but when the email says "look at 16S" and I've got 216 unannotated contigs, I feel like I'm being asked to stir-fry raw timber.

Again, thanks for any help you can provide, and sorry if this sort of question goes against subreddit rules.