Notebook

Quality trim all fastq.gz files using Trimmomatic (v0.30)¶

Code explanation of for loop below:¶

%%bash specifies to use the shell for this Jupyter cell
for file in /Volumes/nightingales/C_gigas/2212_lane2_[^N]* initiates a for loop to handle all files beginning with 2212_lane2_ and only those that do not have the letter "N" at that position in the file name.
do tells the for loop what to do with each of the files.
newname=${file##*/} takes the value of the $file variable (which is /Volumes/nightingales/C_gigas/2212_lane2_[^N]*) and trims the longest match from the beginning of the pattern (the pattern is */; the ## is a bash command to specifiy how to trim). The resulting output (which is just the file name without the full path) is then stored in the newname variable.
This line initiates Trimmomatic and uses the following arguments to specify order of execution:
1. single end reads (SE)
2. number of threads (-threads 16),
3. type of quality score (-phred33),
4. input file location ("$file"),
5. output file name/location (/Volumes/Data/Sam/scratch/20140414_trimmed_$newname),
6. single end Illumina TruSeq adaptor trimming (ILLUMINACLIP:/usr/local/bioinformatics/Trimmomatic-0.30/adapters/TruSeq3-SE.fa:2:30:10),
7. cut number of bases at beginning of read if below quality threshold (LEADING:3)
8. cut number of bases at end of read if below quality threshold (TRAILING:3)
9. cut if average quality within 4 base window falls below 15 (SLIDINGWINDOW:4:15)
done closes for loop.

In [14]:

%%bash
for file in /Volumes/nightingales/C_gigas/2212_lane2_[^N]*
do
newname=${file##*/}
java -jar /usr/local/bioinformatics/Trimmomatic-0.30/trimmomatic-0.30.jar SE -threads 16 -phred33 "$file" /Volumes/Data/Sam/scratch/20140414_trimmed_$newname ILLUMINACLIP:/usr/local/bioinformatics/Trimmomatic-0.30/adapters/TruSeq3-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15;
done

TrimmomaticSE: Started with arguments: -threads 16 -phred33 /Volumes/nightingales/C_gigas/2212_lane2_CTTGTA_L002_R1_001.fastq.gz /Volumes/Data/Sam/scratch/20140414_trimmed_2212_lane2_CTTGTA_L002_R1_001.fastq.gz ILLUMINACLIP:/usr/local/bioinformatics/Trimmomatic-0.30/adapters/TruSeq3-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15
Using Long Clipping Sequence: 'AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA'
Using Long Clipping Sequence: 'AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC'
ILLUMINACLIP: Using 0 prefix pairs, 2 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences
Input Reads: 16000000 Surviving: 15922161 (99.51%) Dropped: 77839 (0.49%)
TrimmomaticSE: Completed successfully
TrimmomaticSE: Started with arguments: -threads 16 -phred33 /Volumes/nightingales/C_gigas/2212_lane2_CTTGTA_L002_R1_002.fastq.gz /Volumes/Data/Sam/scratch/20140414_trimmed_2212_lane2_CTTGTA_L002_R1_002.fastq.gz ILLUMINACLIP:/usr/local/bioinformatics/Trimmomatic-0.30/adapters/TruSeq3-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15
Using Long Clipping Sequence: 'AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA'
Using Long Clipping Sequence: 'AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC'
ILLUMINACLIP: Using 0 prefix pairs, 2 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences
Input Reads: 16000000 Surviving: 15906198 (99.41%) Dropped: 93802 (0.59%)
TrimmomaticSE: Completed successfully
TrimmomaticSE: Started with arguments: -threads 16 -phred33 /Volumes/nightingales/C_gigas/2212_lane2_CTTGTA_L002_R1_003.fastq.gz /Volumes/Data/Sam/scratch/20140414_trimmed_2212_lane2_CTTGTA_L002_R1_003.fastq.gz ILLUMINACLIP:/usr/local/bioinformatics/Trimmomatic-0.30/adapters/TruSeq3-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15
Using Long Clipping Sequence: 'AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA'
Using Long Clipping Sequence: 'AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC'
ILLUMINACLIP: Using 0 prefix pairs, 2 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences
Input Reads: 16000000 Surviving: 15881522 (99.26%) Dropped: 118478 (0.74%)
TrimmomaticSE: Completed successfully
TrimmomaticSE: Started with arguments: -threads 16 -phred33 /Volumes/nightingales/C_gigas/2212_lane2_CTTGTA_L002_R1_004.fastq.gz /Volumes/Data/Sam/scratch/20140414_trimmed_2212_lane2_CTTGTA_L002_R1_004.fastq.gz ILLUMINACLIP:/usr/local/bioinformatics/Trimmomatic-0.30/adapters/TruSeq3-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15
Using Long Clipping Sequence: 'AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA'
Using Long Clipping Sequence: 'AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC'
ILLUMINACLIP: Using 0 prefix pairs, 2 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences
Input Reads: 10634369 Surviving: 10548081 (99.19%) Dropped: 86288 (0.81%)
TrimmomaticSE: Completed successfully
TrimmomaticSE: Started with arguments: -threads 16 -phred33 /Volumes/nightingales/C_gigas/2212_lane2_GCCAAT_L002_R1_001.fastq.gz /Volumes/Data/Sam/scratch/20140414_trimmed_2212_lane2_GCCAAT_L002_R1_001.fastq.gz ILLUMINACLIP:/usr/local/bioinformatics/Trimmomatic-0.30/adapters/TruSeq3-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15
Using Long Clipping Sequence: 'AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA'
Using Long Clipping Sequence: 'AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC'
ILLUMINACLIP: Using 0 prefix pairs, 2 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences
Input Reads: 16000000 Surviving: 15956775 (99.73%) Dropped: 43225 (0.27%)
TrimmomaticSE: Completed successfully
TrimmomaticSE: Started with arguments: -threads 16 -phred33 /Volumes/nightingales/C_gigas/2212_lane2_GCCAAT_L002_R1_002.fastq.gz /Volumes/Data/Sam/scratch/20140414_trimmed_2212_lane2_GCCAAT_L002_R1_002.fastq.gz ILLUMINACLIP:/usr/local/bioinformatics/Trimmomatic-0.30/adapters/TruSeq3-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15
Using Long Clipping Sequence: 'AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA'
Using Long Clipping Sequence: 'AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC'
ILLUMINACLIP: Using 0 prefix pairs, 2 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences
Input Reads: 16000000 Surviving: 15944342 (99.65%) Dropped: 55658 (0.35%)
TrimmomaticSE: Completed successfully
TrimmomaticSE: Started with arguments: -threads 16 -phred33 /Volumes/nightingales/C_gigas/2212_lane2_GCCAAT_L002_R1_003.fastq.gz /Volumes/Data/Sam/scratch/20140414_trimmed_2212_lane2_GCCAAT_L002_R1_003.fastq.gz ILLUMINACLIP:/usr/local/bioinformatics/Trimmomatic-0.30/adapters/TruSeq3-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15
Using Long Clipping Sequence: 'AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA'
Using Long Clipping Sequence: 'AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC'
ILLUMINACLIP: Using 0 prefix pairs, 2 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences
Input Reads: 16000000 Surviving: 15952602 (99.70%) Dropped: 47398 (0.30%)
TrimmomaticSE: Completed successfully
TrimmomaticSE: Started with arguments: -threads 16 -phred33 /Volumes/nightingales/C_gigas/2212_lane2_GCCAAT_L002_R1_004.fastq.gz /Volumes/Data/Sam/scratch/20140414_trimmed_2212_lane2_GCCAAT_L002_R1_004.fastq.gz ILLUMINACLIP:/usr/local/bioinformatics/Trimmomatic-0.30/adapters/TruSeq3-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15
Using Long Clipping Sequence: 'AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA'
Using Long Clipping Sequence: 'AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC'
ILLUMINACLIP: Using 0 prefix pairs, 2 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences
Input Reads: 16000000 Surviving: 15922255 (99.51%) Dropped: 77745 (0.49%)
TrimmomaticSE: Completed successfully
TrimmomaticSE: Started with arguments: -threads 16 -phred33 /Volumes/nightingales/C_gigas/2212_lane2_GCCAAT_L002_R1_005.fastq.gz /Volumes/Data/Sam/scratch/20140414_trimmed_2212_lane2_GCCAAT_L002_R1_005.fastq.gz ILLUMINACLIP:/usr/local/bioinformatics/Trimmomatic-0.30/adapters/TruSeq3-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15
Using Long Clipping Sequence: 'AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA'
Using Long Clipping Sequence: 'AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC'
ILLUMINACLIP: Using 0 prefix pairs, 2 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences
Input Reads: 16000000 Surviving: 15909223 (99.43%) Dropped: 90777 (0.57%)
TrimmomaticSE: Completed successfully
TrimmomaticSE: Started with arguments: -threads 16 -phred33 /Volumes/nightingales/C_gigas/2212_lane2_GCCAAT_L002_R1_006.fastq.gz /Volumes/Data/Sam/scratch/20140414_trimmed_2212_lane2_GCCAAT_L002_R1_006.fastq.gz ILLUMINACLIP:/usr/local/bioinformatics/Trimmomatic-0.30/adapters/TruSeq3-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15
Using Long Clipping Sequence: 'AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA'
Using Long Clipping Sequence: 'AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC'
ILLUMINACLIP: Using 0 prefix pairs, 2 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences
Input Reads: 255678 Surviving: 253470 (99.14%) Dropped: 2208 (0.86%)
TrimmomaticSE: Completed successfully

FASTQC on all trimmed files using FASTQC (v0.11.2)¶

In [16]:

%%bash
for file in /Volumes/Data/Sam/scratch/20150414_trimmed_2212*; do fastqc "$file" --outdir=/Volumes/Eagle/Arabidopsis/; done

Analysis complete for 20150414_trimmed_2212_lane2_CTTGTA_L002_R1_001.fastq.gz
Analysis complete for 20150414_trimmed_2212_lane2_CTTGTA_L002_R1_002.fastq.gz
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Analysis complete for 20150414_trimmed_2212_lane2_GCCAAT_L002_R1_001.fastq.gz
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Analysis complete for 20150414_trimmed_2212_lane2_GCCAAT_L002_R1_006.fastq.gz

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Unzip all FASTQC .zip files¶

In [17]:

cd /Volumes/Eagle/Arabidopsis/

/Volumes/Eagle/Arabidopsis

In [19]:

%%bash
for file in 20150414_trimmed_2212*.zip; do unzip "$file"; done

Archive:  20150414_trimmed_2212_lane2_CTTGTA_L002_R1_001_fastqc.zip
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Archive:  20150414_trimmed_2212_lane2_CTTGTA_L002_R1_002_fastqc.zip
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Archive:  20150414_trimmed_2212_lane2_CTTGTA_L002_R1_003_fastqc.zip
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Archive:  20150414_trimmed_2212_lane2_CTTGTA_L002_R1_004_fastqc.zip
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Archive:  20150414_trimmed_2212_lane2_GCCAAT_L002_R1_001_fastqc.zip
   creating: 20150414_trimmed_2212_lane2_GCCAAT_L002_R1_001_fastqc/
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Archive:  20150414_trimmed_2212_lane2_GCCAAT_L002_R1_002_fastqc.zip
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Archive:  20150414_trimmed_2212_lane2_GCCAAT_L002_R1_003_fastqc.zip
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Archive:  20150414_trimmed_2212_lane2_GCCAAT_L002_R1_004_fastqc.zip
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In [20]:

ls 20150414_*fastqc

20150414_trimmed_2112_lane1_ACAGTG_L001_R1_001_fastqc:
Icons/              fastqc.fo           fastqc_report.html
Images/             fastqc_data.txt     summary.txt

20150414_trimmed_2112_lane1_ACAGTG_L001_R1_002_fastqc:
Icons/              fastqc.fo           fastqc_report.html
Images/             fastqc_data.txt     summary.txt

20150414_trimmed_2112_lane1_ATCACG_L001_R1_001_fastqc:
Icons/              fastqc.fo           fastqc_report.html
Images/             fastqc_data.txt     summary.txt

20150414_trimmed_2112_lane1_ATCACG_L001_R1_002_fastqc:
Icons/              fastqc.fo           fastqc_report.html
Images/             fastqc_data.txt     summary.txt

20150414_trimmed_2112_lane1_ATCACG_L001_R1_003_fastqc:
Icons/              fastqc.fo           fastqc_report.html
Images/             fastqc_data.txt     summary.txt

20150414_trimmed_2112_lane1_CAGATC_L001_R1_001_fastqc:
Icons/              fastqc.fo           fastqc_report.html
Images/             fastqc_data.txt     summary.txt

20150414_trimmed_2112_lane1_CAGATC_L001_R1_002_fastqc:
Icons/              fastqc.fo           fastqc_report.html
Images/             fastqc_data.txt     summary.txt

20150414_trimmed_2112_lane1_CAGATC_L001_R1_003_fastqc:
Icons/              fastqc.fo           fastqc_report.html
Images/             fastqc_data.txt     summary.txt

20150414_trimmed_2112_lane1_GCCAAT_L001_R1_001_fastqc:
Icons/              fastqc.fo           fastqc_report.html
Images/             fastqc_data.txt     summary.txt

20150414_trimmed_2112_lane1_GCCAAT_L001_R1_002_fastqc:
Icons/              fastqc.fo           fastqc_report.html
Images/             fastqc_data.txt     summary.txt

20150414_trimmed_2112_lane1_TGACCA_L001_R1_001_fastqc:
Icons/              fastqc.fo           fastqc_report.html
Images/             fastqc_data.txt     summary.txt

20150414_trimmed_2112_lane1_TTAGGC_L001_R1_001_fastqc:
Icons/              fastqc.fo           fastqc_report.html
Images/             fastqc_data.txt     summary.txt

20150414_trimmed_2112_lane1_TTAGGC_L001_R1_002_fastqc:
Icons/              fastqc.fo           fastqc_report.html
Images/             fastqc_data.txt     summary.txt

20150414_trimmed_2212_lane2_CTTGTA_L002_R1_001_fastqc:
Icons/              fastqc.fo           fastqc_report.html
Images/             fastqc_data.txt     summary.txt

20150414_trimmed_2212_lane2_CTTGTA_L002_R1_002_fastqc:
Icons/              fastqc.fo           fastqc_report.html
Images/             fastqc_data.txt     summary.txt

20150414_trimmed_2212_lane2_CTTGTA_L002_R1_003_fastqc:
Icons/              fastqc.fo           fastqc_report.html
Images/             fastqc_data.txt     summary.txt

20150414_trimmed_2212_lane2_CTTGTA_L002_R1_004_fastqc:
Icons/              fastqc.fo           fastqc_report.html
Images/             fastqc_data.txt     summary.txt

20150414_trimmed_2212_lane2_GCCAAT_L002_R1_001_fastqc:
Icons/              fastqc.fo           fastqc_report.html
Images/             fastqc_data.txt     summary.txt

20150414_trimmed_2212_lane2_GCCAAT_L002_R1_002_fastqc:
Icons/              fastqc.fo           fastqc_report.html
Images/             fastqc_data.txt     summary.txt

20150414_trimmed_2212_lane2_GCCAAT_L002_R1_003_fastqc:
Icons/              fastqc.fo           fastqc_report.html
Images/             fastqc_data.txt     summary.txt

20150414_trimmed_2212_lane2_GCCAAT_L002_R1_004_fastqc:
Icons/              fastqc.fo           fastqc_report.html
Images/             fastqc_data.txt     summary.txt

20150414_trimmed_2212_lane2_GCCAAT_L002_R1_005_fastqc:
Icons/              fastqc.fo           fastqc_report.html
Images/             fastqc_data.txt     summary.txt

20150414_trimmed_2212_lane2_GCCAAT_L002_R1_006_fastqc:
Icons/              fastqc.fo           fastqc_report.html
Images/             fastqc_data.txt     summary.txt

Concatenate two groups of sequences into single file¶

400ppm (control) sequences - Index GCCAAT¶

In [7]:

cd /Volumes/Data/Sam/scratch/

/Volumes/Data/Sam/scratch

In [8]:

%%bash
#gunzips all matching files in folder and appends the data to a single file:
#20150414_trimmed_2212_lane2_400ppm_GCCAAT.fastq
for file in 20150414_trimmed_2212_lane2_G*
do
gunzip -c "$file"  >> 20150414_trimmed_2212_lane2_400ppm_GCCAAT.fastq
done

In [9]:

%%bash
#Gzip file
gzip 20150414_trimmed_2212_lane2_400ppm_GCCAAT.fastq

1000ppm (acidification) sequences - Index CTTGTA¶

In [10]:

%%bash
#gunzips all matching files in folder and appends the data to a single file:
#20150414_trimmed_2212_lane2_1000ppm_CTTGTA.fastq
for file in 20150414_trimmed_2212_lane2_C*
do
gunzip -c "$file" >> 20150414_trimmed_2212_lane2_1000ppm_CTTGTA.fastq
done

In [11]:

%%bash
#Gzip file
gzip 20150414_trimmed_2212_lane2_1000ppm_CTTGTA.fastq

Copy files to Eagle for web-based access¶

In [12]:

%%bash
for file in 2015*e2_[14]*; do cp "$file" /Volumes/Eagle/Arabidopsis/; done