genetica:bioinf_process:fastqc
The evaluation of data quality of the fatsq files is the first one of several Quality Control (QC) steps in the analysis of Next Generation Sequnecing (NGS) data. For such purpose we will use the software FastQC.
The process is quite simple:
- Download and install FastQC in your local server following instructions. In detritus it can be found at
/opt/exoma/bin/fastqc. - Create a directory where to save the outputs of FastQC, for example name it
fastqcRawdata - Check the quality by typing:
$ fastqc -o fastqcRawdata *_1_sequence.fq.gz- the file does not need to be decompressed to run FastQc
- This generates a folder for each file analyzed with several files:
- fastqc_data.txt - this contains the quality statistics in txt format.
- summary.txt - contains a summary of this file quality statistics in form of pass or not pass
- fastqc_report.html - same as before but it can be opened with
$ firefox fastqc_report.htmlwhich allows viewing graphs - Icons - folder with
- Images - folder with graphs as png
To understand the output, there is a nice explanatory video by Babraham Institute.
Example of running FastQC in one of our samples:
Bonn's fastq files are stored at directory: Bonn_0_fastq, under different folders according to its plate of origin, hence:
[vifehe@detritus bonn_data]$ ls Bonn_0_fastq/ P1_001-040 P1_041-080 P1_081-095 P2_001-040 P2_041-080 P2_081-095 P3_001-040 P3_041-080 P3_081-095 P4_001-040 P4_081-095 P4_041-080 P5_001-017
# we create the directory where we will save FastQC output:
[vifehe@detritus Bonn_0_fastq]$ touch fastqcRawdata
# and we further create directories for each of the plates
[vifehe@detritus Bonn_0_fastq]$ cd fastqcRawdata [vifehe@detritus fastqcRawdata]$ touch fastqcRawdata_P1 fastqcRawdata_P2 fastqcRawdata_P3 fastqcRawdata_P4
# to run fastqc on a single file, return to folder where we have our vcf files
[vifehe@detritus fastqcRawdata]$ cd .. [vifehe@detritus Bonn_0_fastq]$ cd P1_001-040 #to see just the first two files [vifehe@detritus P1_001-040]$ ls | head -n2 SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz SN7640211_14074_P1A01_MND1014_2_sequence.fq.gz [vifehe@detritus P1_001-040]$ fastqc -o ../fastqcRawdata/fastqcRawdata_P1 Started analysis of SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz Started analysis of SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz # started at 13:57 Approx 5% complete for SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz Approx 10% complete for SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz Approx 15% complete for SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz Approx 20% complete for SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz Approx 25% complete for SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz Approx 30% complete for SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz Approx 35% complete for SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz Approx 40% complete for SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz Approx 45% complete for SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz Approx 50% complete for SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz Approx 55% complete for SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz Approx 60% complete for SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz Approx 65% complete for SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz Approx 70% complete for SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz Approx 75% complete for SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz Approx 80% complete for SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz Approx 85% complete for SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz Approx 90% complete for SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz Approx 95% complete for SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz Approx 100% complete for SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz Analysis complete for SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz # finished at 14:09 # the process per sample takes about 12 minutes #this can be run in a loop [vifehe@detritus P1_001-040]$ for x in P1_001-040/*.gz; do fastqc -o ../fastqcRawdata/fastqcRawdata_P1/ $x; done # to examine the output [vifehe@detritus P1_001-040]$ cd ../fastqcRawdata/fastqcRawdata_P1 #the program has created a folder named like the sequence and another compressed folder [vifehe@detritus fastqcRawdata_P1]$ ls | head -n2 SN7640211_14074_P1A01_MND1014_1_sequence.fq_fastqc SN7640211_14074_P1A01_MND1014_1_sequence.fq_fastqc.zip #list the contents of the folder created [vifehe@detritus fastqcRawdata_P1]$ cd SN7640211_14074_P1A01_MND1014_1_sequence.fq_fastqc [vifehe@detritus SN7640211_14074_P1A01_MND1014_1_sequence.fq_fastqc]$ ls fastqc_data.txt fastqc_report.html Icons Images summary.txt #examine Summary.txt output [vifehe@detritus SN7640211_14074_P1A01_MND1014_1_sequence.fq_fastqc]$ cat summary.txt PASS Basic Statistics SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz PASS Per base sequence quality SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz PASS Per sequence quality scores SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz PASS Per base sequence content SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz PASS Per base GC content SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz WARN Per sequence GC content SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz PASS Per base N content SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz PASS Sequence Length Distribution SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz WARN Sequence Duplication Levels SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz PASS Overrepresented sequences SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz PASS Kmer Content SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz # examine fastqc_data.txt output [vifehe@detritus SN7640211_14074_P1A01_MND1014_1_sequence.fq_fastqc]$ more -n20 fastqc_data.txt ##FastQC 0.10.1 >>Basic Statistics pass #Measure Value Filename SN7640211_14074_P1A01_MND1014_1_sequence.fq.gz File type Conventional base calls Encoding Sanger / Illumina 1.9 Total Sequences 44012752 Filtered Sequences 0 Sequence length 101 %GC 49 >>END_MODULE >>Per base sequence quality pass #Base Mean Median Lower Quartile Upper Quartile 10th Percentile 90th Percentile 1 31.64506284451379 33.0 31.0 34.0 28.0 34.0 2 31.880190722906853 34.0 31.0 34.0 28.0 34.0 3 31.972653289210363 34.0 31.0 34.0 28.0 34.0 4 35.39369340049448 37.0 35.0 37.0 32.0 37.0 5 35.09201710449735 37.0 35.0 37.0 32.0 37.0 6 35.08697933726116 37.0 35.0 37.0 32.0 37.0 7 35.06162818448617 37.0 35.0 37.0 32.0 37.0 .... .... .... >>Sequence Duplication Levels warn #Total Duplicate Percentage 33.90859348891959 #Duplication Level Relative count 1 100.0 2 29.769972680482393 3 10.634235430848415 4 4.525832792477321 5 1.99009856157457 6 1.1335335758380753 7 0.6905347682369101 8 0.447526904442063 9 0.296590343078804 10++ 1.4482363062804704 >>END_MODULE >>Overrepresented sequences pass >>END_MODULE >>Kmer Content pass >>END_MODULE # Explore html file [vifehe@detritus SN7640211_14074_P1A01_MND1014_1_sequence.fq_fastqc]$ firefox fastqc_report.html # this opens the file in firefox in which the following pictures can be seen
Because examining each file is time consuming, I've created a couple of scripts with which we can extract the information of our interest:
script1 - to summarize output from summary.txt
script2 - to summarize output from fastqc_data.txt
genetica/bioinf_process/fastqc.txt · Last modified: 2023/01/02 15:31 by osotolongo