Error in importRData

[Harshangda Karan Puri]

Hi,

I am getting this error when running the importRData:

Coefficients not estimable: sv28 sv29 sv30 sv31 sv32 sv33 sv34 sv35 sv36 sv37
Coefficients not estimable: sv28 sv29 sv30 sv31 sv32 sv33 sv34 sv35 sv36 sv37
Error in if (m == 0) { : missing value where TRUE/FALSE needed
In addition: Warning messages:
1: Partial NA coefficients for 5153 probe(s)
2: In voom(counts = localDGE, design = localModel, normalize.method = "none",  :
  The experimental design has no replication. Setting weights to 1.

I have created the salmon index by combining GRCm39.primary_assembly.genome.fa.gz and gencode.vM32.transcripts.fa.gz into gentrome.fa.gz file which I used for making Salmon Index. I also used decoy sequences generated from GRCm39.primary_assembly.genome.fa.gz and also used --keepduplicates command. 

I am testing 450 samples belonging to three different conditions and my sample design contains sampleID and condition. 

I am using R4.3, IsoformSwitchAnalyseR 2.01.07 (latest update on GitHub), MacOs Ventura 13.0.1

These are may codes: 

1. For index:

On Command Line:

grep "^>" <(gunzip -c GRCm38.primary_assembly.genome.fa.gz) | cut -d " " -f 1 > decoys.txt

sed -i.bak -e 's/>//g' decoys.txt

cat gencode.vM32.transcripts.fa.gz GRCm39.primary_assembly.genome.fa.gz > gentrome.fa.gz

Job Script for making index:

#!/bin/bash 

#PBS -l nodes=1:ppn=1,mem=40gb 

#PBS -l walltime=01:00:00:00 

#PBS -m abe 

#PBS -M harshangda...@postgrad.manchester.ac.uk 

cd /mnt/panfs1/scratch/wsspaces/hkaranpuri-Alternative-Splicing-0/Salmon2

module load apps/salmon/1.4.0

salmon index -t gentrome.fa.gz -d decoys.txt -p 12 -i salmon_index5 --gencode --keepDuplicates 

2. Job script for quantification: 

fastqDir=/scratch/wsspaces/hkaranpuri-Alternative-Splicing-0/Alternative_Splicing_fastq

SalIndex=/scratch/wsspaces/hkaranpuri-Alternative-Splicing-0/Salmon2/salmon_index5

outDir=/scratch/wsspaces/hkaranpuri-Alternative-Splicing-0/Salmon2/output5

for fn in ${fastqDir}/*R1*.fastq.gz ;

do

                noExt=${fn%%_R1_001.fastq.gz}

                samp=`basename ${noExt}`

                inputFastq2=${noExt}_R2_001.fastq.gz

                dircur=`dirname ${fn}`

qsub -V <<-_EOF

#!/bin/bash

#PBS -l nodes=2:ppn=8

#PBS -l mem=30gb

#PBS -l walltime=01:00:00:00

#PBS -N SalmonQuant_${noExt}

cd $PBS_O_WORKDIR/

#module load apps/salmon/0.13.0

module load apps/salmon/1.2.0

echo 'Processing sample ${samp}'

echo 'Fastq1 ${fn}'

echo 'Fastq2 ${inputFastq2}'

salmon quant -i ${SalIndex} -l A \

         -1 ${fn} \

         -2 ${inputFastq2} \

         -p 8 --validateMappings -o ${outDir}/${samp}_quant

_EOF

sleep 0.2

[Kristoffer Vitting-Seerup]

Please don't cross-post problems. I answered on GitHub.