6. 16S Analysis

Preprocessing (demux) with qiime

on atlantis

docker run -it --rm -v $(pwd):$(pwd) -w $(pwd) quay.io/biocontainers/qiime:1.9.1--py_3 /bin/bash

1. Create mapping files: (mapping.txt)

#SampleID BarcodeSequence LinkerPrimerSequence Description Presti_H2O_A TCCCTTGTCTCC Rachel_Presti Presti_17681 ACGAGACTGATT Rachel_Presti

2. Validate mapping file:

validate_mapping_file.py -m /scratch/sahlab/MiSeq_M317/MiSeq_M317_mapping.txt

Errors and/or warnings detected in mapping file.

MiSeq_M317_mapping_corrected.txt

NOTE: Try using the corrected file for the next step

3. Extract the single-end barcodes from the I1 file:

extract_barcodes.py --input_type barcode_single_end -f /path/to/M557_S0_L001_I1_001.fastq.gz --bc1_len 12 -o /path/to/barcode/output/file

NOTE: --input_type barcode_single_end because only I1, no I2

4. Split the R1 files:

split_libraries_fastq.py -i /path/to/M557_S0_L001_R2_001.fastq.gz -b /path/to/barcode/barcodes.fastq -m /path/to/mappingFile --barcode_type golay_12 --rev_comp_mapping_barcode --store_demultiplexed_fastq -r 999 -n 999 -q 0 -p 0.001 -o /path/to/demultiplexed/files

If you get this error message:

Some or all barcodes are not valid golay codes. Do they need to be reverse complemented?

Add --rev_comp_mapping_barcode:

split_libraries_fastq.py -i /path/to/M557_S0_L001_R1_001.fastq.gz -b /path/to/barcode/barcodes.fastq -m /path/to/mappingFile --barcode_type golay_12 --rev_comp_mapping_barcode --store_demultiplexed_fastq -r 999 -n 999 -q 0 -p 0.001 -o /path/to/demultiplexed/files

5. Split the R1 files out of seqs.fastq:

split_sequence_file_on_sample_ids.py -i /path/to/seqs.fastq -o /path/to/split/files --file_type fastq

5b. Add R1 to the fastq names:

rename .fastq _R1.fastq *.fastq

mv barcodes_R1.fastq barcodes.fastq
mv seqs_R1.fastq seqs.fastq
mv reads_R1.fastq reads.fastq

6. Split the R2 files:

split_libraries_fastq.py -i /path/to/M557_S0_L001_R1_001.fastq.gz -b /path/to/barcode/output/file/barcodes.fastq -m /path/to/mappingFile --barcode_type golay_12 --rev_comp_mapping_barcode --store_demultiplexed_fastq -r 999 -n 999 -q 0 -p 0.001 -o /path/to/demultiplexed/files

7. Split the R2 files out of seqs.fastq:

split_sequence_file_on_sample_ids.py -i /path/to/seqs.fastq -o /path/to/split/files --file_type fastq

7b. Add R1 to the fastq names:

cd R2
rename .fastq _R1.fastq *.fastq

mv barcodes_R2.fastq barcodes.fastq
mv seqs_R2.fastq seqs.fastq
mv reads_R2.fastq reads.fastq

DADA2