I am a complete rookie in shotgun analysis, having only performed marker gene analysis before. I have a pipeline that I want to try but I can't wrap my head around how it all works.
Please correct me if I'm wrong:
I start with a paired-end Illumina short reads fastq files (.R1,.R2)
I will use FASTQC to trim according to quality. I assume that since data has been demultiplexed, the primers and and adaptors are removed as well.
? First question, a lot of people advise to merge all the samples together and then do the co-assembly since apparently it helps with the assembly. Would this be necessary if I also have nanopore reads and only 9 samples? If if I did this, how do I then separate the samples from each other?
Because I also have nanopore, I will use a hybrid assembler to get contigs. The output will be a file with contigs.
I then plan to use a binning tool (Metawrap) to create bins.
? I believe the output of these will be a file containing bins per sample. How do I estimate the abundance of each bin in the sample? And how do I factor in the depth of sequencing to normalise them? This is my biggest problem, I can't wrap my head around it.
?Can some explain VERY plainly what is sequencing depth and how do I factor it into my analysis? I've seen some people using it for normalisation of the bin/contigs abundance but again, this is idea is not intuitive to me at all.
- From then on, taxonomy and functional annotation is pretty straightforward.
So a little background on the project. I have samples sequenced from a bioreactor containing a small community of bacteria (no host removal required). The samples derive from 6 different reactors = 6 samples as well as one reactor will be samples three times (time series) so I have 9 samples in total. The shotgun Illumina will be combined with nanopore. The idea is to get the composition of the community (what kind of bacteria are there?), abundance (what is their relative quantity?) but also functions (what do they do?).
I'm sorry for a very generic question, I would really appreciate somebody answering very plainly without too much jargon. Hopefully it will help me and other people who are just starting out with the shotgun.