Hello,
I am following Miseq SOP. I need to quality filter my data using qwindowaverage and qaverage in trim.seq command line. For this, I will prepare qual file using fastq.info.
But I am confused that at which step I should prepare qual files, before making contigs or after that ? Also, should I make qual files for all forward and reverse reads SEPERATELY ??? ; and then write names of all the qual files in a notepad which can be used later at trim.seqs command? If yes, what should be the format in the notepad ?
If NO, do I have to make a file in notepad (in which names of the samples and their respective fastq files i.e, of forward and reverse reads is written) as we do for make.contigs command (i.e, preparing stability.file ???) and then select that file for doing fastq.info ?
Doing this Trim.seqs will from completely throw out the sequence (contig) from analyses ?
I tried to start in this way- I made a file of notepad in this way (for 3 samples) -
sample1 J1_1.fastq J1_2.fastq
sample2 J2_1.fastq J2_2.fastq
sample3 J3_1.fastq J3_2.fastq
Then I saved it as data.fastq.
Then the steps that I planned to follow was
mothur> fastq.info(fastq=data.fastq)
mothur> make.contigs(file=stability.files)
mothur > trim.seqs(fasta=Data.fasta, qfile=Data.qual, qaverage=28), nd then so on …
But I got following message after the first step-
mothur > fastq.info(fastq=data.fastq, processors=4)
processors is not a valid parameter.
The valid parameters are: fastq, oligos, group, pdiffs, bdiffs, ldiffs, sdiffs, tdiffs, fasta, qfile, pacbio, format, inputdir, and outputdir.
[ERROR]: did not complete fastq.info.
mothur > fastq.info(fastq=data.fastq)
[WARNING]: reading GON1 expected a name with @ as a leading character, ignoring read.
[WARNING]: reading GON28 expected a name with + as a leading character, ignoring.[WARNING]: missing quality for GON28, ignoring.[WARNING]: names do not match. read GON1 for fasta and GON28 for quality, ignoring.[WARNING]: Lengths do not match for sequence GON1. Read 26 characters for fasta and 0 characters for quality scores, ignoring read.
Please suggest, how should I design the steps ? Thanks to all of you who helped me so far. I am sorry if I asked a very basic question Looking forward, Richa