Salmon building index and quantifying mapping questions

Question

[slin023]

Hi, Rob I have worked done De Novo assembly to build a transcriptome file "Phormia_de_novo_transcriptome_assembly_V1.fasta". If I would like to assess the quality by mapping the raw read "00_fastq.txt" to my assembly file "Phormia_de_novo_transcriptome_assembly_V1.fasta". I know the first thing is to build the index, which I have tried salmon index -t /Users/maysonlin/Downloads/De_Novo/Phormia_de_novo_transcriptome_assembly_V1.fa -i /Users/maysonlin/Downloads/De_Novo/Phormia_de_novo_transcriptome_assembly_V1.index -- type quasi -k 31 , I also looked up for some Youtube videos, so far didn't get anywhere, I have been stuck for many days. Does the assembly file and raw read file have to be in the same folder in Salmon files? What command line should I build? Please let me know, I sincerely appreciate it! Best Mayson

[jared.andrews07]

Are you getting an error message?

[slin023]

Kinda of, it shows "command not found "

[Rob]

It sounds like the salmon executable is not in your path. What do you get if you simply write:

which salmon

at your command prompt? How did you install salmon?

[slin023]

it shows

/opt/anaconda3/envs/salmon/bin/salmon I installed it via bioconda

[Rob]

In your command, you have a space between -- and type. There shoild be no space there. Also, for recent versions of salmon (1.0.0 and later), there is no quasi index type. The new index type is called puff, but you can leave off the --type argument all together and just try:

salmon index -t /Users/maysonlin/Downloads/De_Novo/Phormia_de_novo_transcriptome_assembly_V1.fa -i /Users/maysonlin/Downloads/De_Novo/Phormia_de_novo_transcriptome_assembly_V1.index

[slin023]

Oh my god, thank you so much, it took me more than a week to just build an index. However, I just realized based on postdoc assigning task for me is "2. FASTQ quality trims your reads." by trimmomatic , do I just download the trimmomatic files to any directory or specific directory to make it work? or if I can download trimmomatic via bioconda? will it work with Salmon?

[jared.andrews07]

That step would be performed before Salmon. The resulting FASTQ files will work fine with Salmon. You can install it manually or via bioconda - it should work the same regardless. bioconda will add it to your PATH automatically for the virtual environment, so that's probably the easier method.

It sounds like it may be worth your time to see if said post-doc will sit down with you for a few minutes and explain each step and why each is necessary.

[slin023]

I have trimmomatic installed and I have read the manual. The only I am concerned about is giving input files for "paired-end" mode in trimmomatic command lines. I followed up the script and revised it. java -jar /opt/anaconda3/pkgs/trimmomatic-0.39-1/share/trimmomatic-0.39-1/trimmomatic.jar -threads 16 -phred33 ${SRR}_1.fastq.gz ${SRR}_2.fastq.gz ${SRR}_1_paired.fastq.gz ${SRR}_1_unpaired.fastq.gz ${SRR}_2_paired.fastq.gz ${SRR}_2_unpaired.fastq.gz ILLUMINACLIP:/opt/anaconda3/pkgs/trimmomatic-0.39-1/share/trimmomatic-0.39-1/adapters/TruSeq3-PE-2.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36 so for example, if I would like to put raw read data 00_fastq.text, I change ${SRR}_1.fastq.gz into /Users/maysonlin/Downloads/PhormiaMaleFemaleTranscriptome_Stranded/Phormia raw data/00_fastq/00_1.fastq.qz, do I follow the right track?

I tried to revise the directory and file names, it gives this

Usage: PE [-version] [-threads <threads>] [-phred33|-phred64] [-trimlog <trimLogFile>] [-summary <statsSummaryFile>] [-quiet] [-validatePairs] [-basein <inputBase> | <inputFile1> <inputFile2>] [-baseout <outputBase> | <outputFile1P> <outputFile1U> <outputFile2P> <outputFile2U>] <trimmer1>... or: SE [-version] [-threads <threads>] [-phred33|-phred64] [-trimlog <trimLogFile>] [-summary <statsSummaryFile>] [-quiet] <inputFile> <outputFile> <trimmer1>... or: -version