De-multiplexed FASTQ

Description

The next steps assume that your data comes back from the sequencing facility with a FASTQ file for each sample. Before we can generate any OTU's or microbiome composition counts, the sequencing reads must first be joined, filtered and de-multiplexed. The following sections begin to outline this plan.

1. Create a qiime parameters file

2. Multiple Join Paired Ends

http:\/\/qiime.org\/scripts\/multiple_join_paired_ends.html

Description

Parameters

--forward_reads_fp | -f
The forward illumina fastq read. Typically will have the characters R1

--reverse_reads_fp | -r
The reverse illumina fastq read. Typically will have the characters, R3

--index_reads_fp | -b
The barcode illumina fastq read. Typically will have the characters, `R2

--output_dir | -o
The name of the folder to place output files.

--pe_join_method | -m
Which program to use for joining reads. Can be either fastq-join or SeqPrep

--min_overlap | -j
The minimum number of overlapping base pairs between the two reads. (This is an important parameter depending upon the length of your reads. In a 150x150 Paired-End read run there should be a 50bp overlap when the two reads are merged. Increasing this number increases the stringency of your sequence filtering and you will lose low quality scores. Please consider what this number should be based on your data!)

--perc_max_diff | -p
Maximum allowed % differences within region of overlap.

Command

# Multiple joining of paired ends
multiple_join_paired_ends.py \
-i TG_Microbiome_Dataset_April_2016 \
-o join_paired_ends \
-p qiime_parameters.txt

Output

(TODO)

3. Multiple Split Libraries

http:\/\/qiime.org\/scripts\/multiple_split_libraries_fastq.html

Description

(TODO)

Parameters

--forward_reads_fp | -f
The forward illumina fastq read. Typically will have the characters R1

--reverse_reads_fp | -r
The reverse illumina fastq read. Typically will have the characters, R3

--index_reads_fp | -b
The barcode illumina fastq read. Typically will have the characters, `R2

--output_dir | -o
The name of the folder to place output files.

Command

multiple_split_libraries_fastq.py \
-i join_paired_ends \
-o split_libraries \
--demultiplexing_method sampleid_by_file \
-p qiime_parameters.txt

Output