Sequencing Results

Sequencing Results

Mock-Community

MiSeq Quality scores

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Sequence Results

Soil Column I

MiSeq Quality scores

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Sequence Results

Soil Column II

MiSeq Quality scores

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Sequence Results

Swine Fecal

MiSeq Quality scores

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Sequence Results

Read Processing Pipeline

Workflow

*Demultiplexing done for analysis, though not necessarily required for practical implementation of DARTE-QM.

Software

Cutadapt - demultiplexing and primer-trimming
Cutadapt removes adapter sequences from high-throughput sequencing reads

PEAR - paired-end read assembler
PEAR: a fast and accurate Illumina Paired-End reAd mergeR

BLAST - basic local alignment search tool
Basic local alignment search tool

RDP-Classifier - 16S classifier
Naïve Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy

Primer Trimming and Demultiplexing

wd="../data/read_processing"
raw_files="${wd}/raw"

for raw_file in $(ls ${raw_files}/putvariablehere*_R1_*)
do \
sample="$(basename -- $raw_file)"
sample=$(echo "$sample" | cut -f 1 -d '.')
sample=$(echo "$sample" | cut -f 1 -d '_')

mkdir -p ${wd}/split/${sample}

cutadapt \
    --pair-filter=both \
    -e 0.10 \
    -q 15 \
    -g file:${wd}/../data/DARTE_forward_primers \
    -G file:${wd}/../data/DARTE_reverse_primers \
    -o ${wd}/split/${sample}/{name}.R1.1.fastq \
    -p ${wd}/split/${sample}/{name}.R2.1.fastq \
    $(ls ${wd}/raw/${sample}*_R2_*) \
    $(ls ${wd}/raw/${sample}*_R1_*)
cutadapt \
    --pair-filter=both \
    -e 0.10 \
    -q 15 \
    -g file:${wd}/../data/DARTE_forward_primers \
    -G file:${wd}/../data/DARTE_reverse_primers \
    -o ${wd}/split/${sample}/{name}.R1.2.fastq \
    -p ${wd}/split/${sample}/{name}.R2.2.fastq \
    ${wd}/split/${sample}/unknown.R2.1.fastq \
    ${wd}/split/${sample}/unknown.R1.1.fastq
done

Pair-end Read Merging

for primer in $(grep ">" ${wd}/../data/DARTE_forward_primers  | cut -c2-)
do \
mkdir -p ${wd}/merge/${sample}
mkdir -p ${wd}/combined/${sample}

cat ${wd}/split/${sample}/${primer}*R1* > ${wd}/split/${sample}/${primer}.R1.fastq
cat ${wd}/split/${sample}/${primer}*R2* > ${wd}/split/${sample}/${primer}.R2.fastq

for file in $(ls ${wd}/split/${sample}/*.R1.fastq)
do \
file_name="$(basename -- $file)"
file_name=${file_name%.fastq}
file_name=$(echo $file_name| tr : _)
file_name=$(echo "$file_name" | cut -f 1 -d '.')

pear/bin/pear \
-j 8 \
-p 0.05 \
-v 10 \
-m 350 \
-f ${file} \
-r ${wd}/split/${sample}/${file_name}.R2.fastq \
-o ${wd}/merge/${sample}/${file_name}_merged

cat ${wd}/merge/${sample}/${file_name}_merged.assembled.fastq \
    ${wd}/merge/${sample}/${file_name}_merged.unassembled.forward.fastq \
    ${wd}/merge/${sample}/${file_name}_merged.unassembled.reverse.fastq \
    > ${wd}/combined/${sample}/${file_name}_combined.fastq;

sed -n '1~4s/^@/>/p;2~4p' ${wd}/combined/${sample}/${file_name}_combined.fastq \
    > ${wd}/combined/${sample}/${file_name}.fasta
done
done

BLAST

makeblastdb -in ../data/ARG_database.fa -dbtype nucl -out ../data/DARTE-QM_ARGs

for primer in $(grep ">" ${wd}/../data/DARTE_forward_primers  | cut -c2-)
do
cat ${wd}/split/${sample}/${primer}*R1* > ${wd}/split/${sample}/${primer}.R1.fastq
cat ${wd}/split/${sample}/${primer}*R2* > ${wd}/split/${sample}/${primer}.R2.fastq

for file in $(ls ${wd}/split/${sample}/*.R1.fastq)
do \
file_name="$(basename -- $file)"
file_name=${file_name%.fastq}
file_name=$(echo $file_name| tr : _)
file_name=$(echo "$file_name" | cut -f 1 -d '.')

/mnt/home/smithsch/software/ncbi-blast-2.9.0+/bin/blastn \
    -db ../data/DARTE-QM_ARGs \
    -query ${wd}/combined/${sample}/${file_name}.fasta \
    -out ${wd}/blast/${sample}/${file_name}.blast \
    -perc_identity 90 \
    -outfmt 6 \
    -num_threads 8;

done
done

Schuyler Smith
Ph.D. Student - Bioinformatics and Computational Biology
Iowa State University. Ames, IA.