New to qiime - getting started question

[IDA]

Hi all, 

I am completely new to qiime and I am currently running through the 454 Overview tutorial, however I have run into a problem already... 

When trying to validate my mapping file which comes in the qiime_tutorial folder dataset this error message occurs. 

MacQIIME Idas-MacBook-Pro:~ $ cd qiime_tutorial

MacQIIME Idas-MacBook-Pro:qiime_tutorial $ validate_mapping_file.py -m Fasting_Map.txt -o mapping_output

Error in validate_mapping_file.py: option -m: file does not exist: 'Fasting_Map.txt'

Have I simply saved this folder in the wrong place? Sorry for a pretty silly question but as I said, just started to try to get my head around this... 

Thank you, 

Ida 

[IDA]

When I ask to list the files in the tutorial folder it list everything except the .txt files 

MacQIIME Idas-MacBook-Pro:~ $ cd qiime_tutorial

MacQIIME Idas-MacBook-Pro:qiime_tutorial $ ls

18S_tutorial_files   Fasting_Example.qual

Fasting_Example.fna  Fasting_Example.sff

MacQIIME Idas-MacBook-Pro:qiime_tutorial $ 

[justink]

That's strange. I'd say delete the qiime_tutorial folder and download again from here: http://qiime.org/tutorials/tutorial.html#obtaining-the-data .

Also for me it unzips to the name: 'qiime_overview_tutorial' . I wonder if that's related.

[IDA]

Hi there @justink

Thank you, I deleted the folder and downloaded it again and it works just fine! :) 

I have now run through the whole tutorial data set and created my tables, trees and graphs and its all working!! :) 

I have a real data set though, which is information from an excel sheet. I have converted it to .txt and validated my mapping file (and corrected warnings and errors and its good to go now). 

But I am finding it hard to find anywhere which tells me what to do next... 

So I have a lot of files which I have downloaded from base space and a valid mapping file. I have started by unzipping the base space .fastq.tz files and now they are .fastq files. 

Whats the next step? 

Thankful for help!! :D 

All the best, 

Ida 

[Jotham Suez]

Hi Ida,

This will of course vary depending on how you prepared your experiment and the run. Very generally the flow will be:

0. fastqc

1. join_paired_ends

2. split_libraries_fastq

3. pick_closed_reference_otus / pick_open_reference_otus

After this step you will have a BIOM table which is used for all the downstream analysis, such as

summarize_taxa_through_plots

beta_diversity_through_plots

make_distance_boxplots

alpha_rarefaction

It is really helpful to read what every script does and what are the required inputs and outputs, there's a dedicated page for each one. I will be able to give more details later if you need them. Good luck!

[justink]

Agreed. Split_libraries_fastq is likely to be the tricky bit—but it sounds like (perhaps?) each .fastq file is its own sample, and there's options for that. Let us know if you're having trouble getting past the split_libraries_fastq stage, and include an example command, yeah?

[IDA]

Hi @justink and @Jotham Suez

I have another question...

I have a R1 and R2 .fastq file of each of my samples. 

Therefore, before I ran the multiple_split_libraries_fastq.py I ran the command multiple_join_paired_ends.py see below: 

MacQIIME Idas-MacBook-Pro:~ $ multiple_join_paired_ends.py -i /users/idacarolinelundbeck/Fastq_files_YBG -o /users/idacarolinelundbeck/Fastq_files_YBG/joined

This gives me a folder for each sample which contains 3 files: Fastq.join.fastq, Fastq.un1.fastq and Fastq.un2.fastq!

I am not too sure of which of these files to use? 

I tried to run the multiple_split_libraries_fastq.py just right after on the files in the 'jones' folder and it worked fine to run... 

however it was not until after I realised that the analysis then might be on unpaired ones, particularly form the reverse reads that are kept and included? Would I need to move all files to a new folder, delete un1 and un2 and re-run ultiple_split_libraries_fastq.py on just the Fastq.join.fastq files? 

Thanks again for all the help! :) 

[justink]

Sorry for the slow reply—was off kayaking. Yes, the 'join' file contains those reads that were successfully joined, un1 contains the seqs from the forward reads that didn't join, un2 from the reverse reads. Hopefully most of your reads fall in the 'joined' file, and that's the one to use. I typically ignore the unjoined reads if they're a small portion of the total.