I have 12 different RNA-seq metatranscriptome samples that I am trying to assemble using Trinity, but my computer (and Galaxy) cannot handle the size of the samples. The sample list is as follows: Microbe type #1 control Microbe type #1 test condition Microbe type #2 control Microbe type #2 test condition Microbe type #3 (from culture #1) control Microbe type #3 (from culture #1) test condition Microbe type #3 (from culture #2) control Microbe type #3 (from culture #2) test condition Microbe type #4 (from culture #3) control Microbe type #4 (from culture #3) test condition Microbe type #4 (from culture #4) control Microbe type #4 (from culture #4) test condition
Microbe type #3 is a specific category of bacteria, but culture #1 and culture #2 consist of different microbial populations. This goes for microbe type #4 too, with culture #3 and culture #4 consisting of different microbial populations.
These 12 samples were grown in 12 different bioreactors, so there are no replicates (the RNA was extracted and sequenced only once, resulting in 12 different paired-end fastq files).
I have tried putting all 24 fastq files into the same Trinity command:
Trinity --seqType fq --max_memory 200G \
--left sample1.1.fastq.gz,sample2.1.fastq.gz, ... sample12.1.fastq.gz \
--right sample1.2.fastq.gz,sample2.2.fastq.gz, ... sample12.2.fastq.gz --CPU 64
However, this causes my computer to run out of storage and memory. For my purposes, would it be okay to assemble each of the 12 samples one at a time? Alternatively, what if I did 2 assembly runs, with one run consisting of Microbe type #1 and Microbe type #2 (4 reads total), and the second run consisting of Microbe type #3 and Microbe type #4 (8 reads total)?
How would this affect the downstream analyses for quantifying the transcripts and gene abundances, and for differential expression analysis? I know that not having replicates isn't good for analysis but the experimental design wasn't planned well, and I'm not able to re-extract RNA or get more sequencing information. Any advice would be greatly apppreciated.
