Hi mothur members. I am new to mothur and i am curious to know that in Miseq SOP they aligned their sequence in a particular region of silva database. But how do i know by aligning in which region i will get good results. Is there any criteria to know how to align your sequence in a specific region.
seq.error
One more doubt they are checking their error rates by using a mock group. How can i make a mock group for my sequence file?
last doubt
make.contigs
Sometimes after running make.contigs command for pair-end reads it will giving Group, fasta, report file but not giving count_table file. so after running uniqe.seqs command it will generating a .names file by using .names file and previously generated .groups file i am running count.seqs to get a count_table. Is it right way to get count_table or there is any other way to get count_table after running make.contigs command?
If you follow the MISEQ tutorial, you should get your answer.
You can use PCR.seq but with the option oligo instead of the region. That will give you a file for alignment tailored to your needs. Or you can align all your data on the complete database and using summary you will see exactly where most of your sequences are.
Seq.error:
This is for setting the error rate and for this you need a positive control. Normally, you know the sequences you used for the positive control (home made or commercial solution). Just use the unaligned option. A moc group is not made by bioinformatics, it is made as a sample you send with yours to sequencing. It cannot be made post experimentation.It is a control, like a positive control you would use for ELISA or PCR.
For the make.contigs, download the latest version of Mothur because all the SOP online has been adapted to 1.47 where HUDGE changes were made. You will have to rewrite some of you “old” commands to fit with the new SOP.
In any case, I suggest that for all commands you use in Mothur that you go read more on them. You can click on the command as you read the SOP.
