BOL

Once your assembly is complete, there are several analyses you will likely want to pursue to explore aspects of the biology of your organism based on your assembled transcripts and the input RNA-Seq data. Such studies might include:

• Quantifying transcript and gene abundance. This is a prerequisite to many other analyses such as examining differentially expressed transcripts among your samples.

• Quality checking your samples and biological replicates. Make sure the relationships among your samples and biological replicates make sense. If there are any confounding aspects to the data, such as outliers or batch effects, you'll want to catch them as early as possible and account for them in your further data explorations.

• Perform differential expression analysis. Trinity provides direct support for several DE analysis methods, including edgeR, DESeq2, Limma/Voom, and ROTS.

• Extract coding regions using TransDecoder and functionally annotate the transcripts using Trinotate.

• If your organism has an assembled genome, consider using the Trinity transcriptome assemblies for gene structure annotation using PASA.

Using KAT again (You will need the modules: KAT/2.1.1 and gnuplot/4.6.5) – we can plot the kmer content of the assembly compared to the kmer content of the read set. The first thing we need to do is to combine the reads into a single file, for gzipped files, this can be done with zcat, or for unzipped files cat.

Ex.

$ cat reads_R1.fastq >> combined.fastq
$ zcat reads_R2.fastq.gz >> combined.fastq

We will now use kat comp to create a kmer content comparison. Use kat comp --help to get help for the program, then create a comparison between the combined reads and the assembly. Make sure that you use the flags for canonical hashes for both sequence 1 and 2, as well as 8 threads. Finally, clean up you working directory by removing the combined fasta file, and re-zipping any unzipped files. Then download the output files to you computer using scp and look at the png file that was produced.

• Does the kmer content look good to you?
• How much of the kmer “noise” is part of the final assembly?
• What do you think contamination would look like in the kmer plot?
• What can the kmer graph tell you about the ploidy of the organism?

Sometime, you need to provide the path 

➜ Tools git:(master) ✗ PATH=$PATH:/home/tools/anaconda3/bin 
➜ Tools git:(master) ✗ export $PATH