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3 hours ago by

Dear All

I had RNAseq data from different plant tissues (eg, root, leave, fruit). I managed to perform de novo assembly using Trinity. Then, I proceed with TGICL to obtain Unigenes.

In order to obtain gene expression value, I used both RSEM and salmon.

Example of script for RSEM

rsem-prepare-reference Unigenes.fasta Unigenes.fasta
rsem-calculate-expression --bowtie2 -p 20 --paired-end SRR5904767_1.trimmed.fastq.gz SRR5904767_2.trimmed.fastq.gz Unigenes.fasta R_A1

Example of Script for salmon

salmon index -t Unigenes.fasta -i unigenes_index 
perl batch.pl Trimmed_data_set

batch,pl file

#!usr/bin/perl
use autodie;
open FILE, "$ARGV[0]", or die;
while (<FILE>) {
        chomp $_;
        @column = split (/t/, $_);
    #   print "$column[1]tt$column[2]tt$column[3]n";
        print "Running $column[1] and $column[2] against unigenes_index. Output to quants/$column[1]n";
        system ("salmon quant -i unigenes_index -l A -1 $column[2] -2 $column[3] -p 20 --validateMappings -o quants/$column[1]");
        }
  1. I noticed that RSEM produced both TPM and FPKM value whereas salmon produced TPM and NumReads. However, TPM value produced from RSEM and salmon is a bit different. May I know which value (FPKM from RSEM or TPM from salmon or RSEM) should I proceed with Differential expression analysis? If TPM to use, what can we do to the FPKM value?

According to the paper in the link below, it mentioned that
normalization metrics should be avoided as RPKM has shown to be inconsistent and Transcripts Per Million (TPM) is preferable
www.intechopen.com/books/applications-of-rna-seq-and-omics-strategies-from-microorganisms-to-human-health/rna-seq-applications-and-best-practices

  1. Should I performed normalization first before DE analysis or not needed as normalization already done in salmon?

Any paper suggestion for step by step analysis if I would like to do differential gene expression using the Unigenes instead of the transcript?

The main objective is to get the differential expressed genes in fruit during 3 different time points and I have two biological replicate only.

Thanks

link

modified 2 hours ago

written
3 hours ago
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wes10



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