Hi guys, I need your kind support:)
I am doing de novo RNA-seq analysis by using Trinity. At first I combined left reads and right leads, which were obtained from three different conditions.
cat right1.fastq right2.fastq right3.fastq > R1.fastq
cat left1.fastq left2.fastq left3.fastq > R2.fastq
And I assembled these reads by Trinity to produce assembled Fasta file (Trinity.fasta).
Trinity --seqType fq --right R1.fastq --left R2.fastq --SS_lib_type RF -min_contig_length 500--CPU 8 --max_memory 16G
Finally, I mapped the individual reads to the created Trinity.fasta. The below code shows the mapping of only right1.fastq and left1.fastq, but I did the same thing for the remaining fastq files.
align_and_estimate_abundance.pl --right1.fastq --left left1.fastq --est_method RSEM --aln_method bowtie2 --trinity_mode --prep_reference --output_dir ./output_files --transcripts Trinity.fasta
However, looking at the number of hits in the three RSEM.isoforms.results, there were many Trinity reads that were not hit under any conditions. Since the Trinity reads were constructed by the fastq data, I think it is very strange that no hits were created.
Are there any possible reasons for this phenomenon?