Quality Control and Preprocessing Exercise - Revision history

Rf at 14:05, 14 May 2017

2017-05-14T14:05:38Z

Rf at 11:33, 9 May 2017

2017-05-09T11:33:11Z

Rf at 11:31, 9 May 2017

2017-05-09T11:31:07Z

Rf at 15:02, 8 May 2017

2017-05-08T15:02:10Z

Rf at 15:35, 5 May 2017

2017-05-05T15:35:16Z

Rf at 15:33, 5 May 2017

2017-05-05T15:33:36Z

Rf at 14:05, 5 May 2017

2017-05-05T14:05:19Z

Rf at 13:30, 5 May 2017

2017-05-05T13:30:18Z

Rf at 11:48, 5 May 2017

2017-05-05T11:48:13Z

Rf: Rf moved page Quality Control and Processing Exercise to Quality Control and Preprocessing Exercise

2017-05-04T13:00:07Z

Rf moved page Quality Control and Processing Exercise to Quality Control and Preprocessing Exercise

@@ Line 22: / Line 22: @@
 = View data set =
-  cd $HOME/i2rda_data/Quality_Control_and_Preprocessing
+First we go into the appropriate directory:
   zcat Read_1.fastq.gz |head
   zcat Read_2.fastq.gz |head

@@ Line 76: / Line 76: @@
 * Did qualities improve?
-A custom utility such as fqzinfo can give succint information about fastq.gz reads, to understand its output, type
+A custom utility such as <code>fqzinfo</code> can give succint information about <code>fastq.gz</code> reads, to understand its output, type
   fqzinfo
-Then run it again specifying the fastq.gz files you are interested in. Or, try all of them (will tkae longer of course):
+Then run it again, this time specifying the fastq.gz files you are interested in. Or, try all of them (will take longer of course):
   fqzinfo *.fastq.gz
 * Did we lose much raw data in this clipping process?

@@ Line 76: / Line 76: @@
 * Did qualities improve?
-A custom utility such as fqzinfo can give succint information about fastq.gz reads
+A custom utility such as fqzinfo can give succint information about fastq.gz reads, to understand its output, type
-  fqzinfo Read_1_q32l50.fastq.gz Read_2_q32l50.fastq.gz
+  fqzinfo
 * Did we lose much raw data in this clipping process?

@@ Line 84: / Line 84: @@
 = If you have time to spare =
-* Run fastq-mcf again but this time using a quality threshold of 30
+* Run fastq-mcf again but this time using a differnt quality threshold, say 28.
-* It may be the reduction in quality is small, but that many more reads and bases are retained.
+* Run FastQC on the new fastq files and then use multiqc to compare your unfiltered and two alternatively filtered fastq pairs.

← Older revision		Revision as of 15:35, 5 May 2017
Line 85:		Line 85:

	* Run fastq-mcf again but this time using a quality threshold of 30		* Run fastq-mcf again but this time using a quality threshold of 30
		+	* It may be the reduction in quality is small, but that many more reads and bases are retained.

@@ Line 58: / Line 58: @@
 * -q quality threshold causing base removal
 * -l Minimum remaining sequence length
-* --qual-mean - Minimum mean quality score
+* --qual-mean - Minimum mean quality score, taking the other pair into account
 As you can see fastq-mcf is able to deal with multiple files in the one command.

@@ Line 14: / Line 14: @@
 * FastQC: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/
-* FastqMcf: https://code.google.com/p/ea-utils/wiki/FastqMcf
+* Fastq-mcf, part of the ea-utils suite: https://code.google.com/p/ea-utils/wiki/FastqMcf
   module load FASTQC ea-utils
@@ Line 28: / Line 28: @@
 where:
 * zcat outputs gzip-compress files to the screen
-* | is the piple operator takes output, converts it to input
+* | is the pipe operator which takes output, converts it to input
 * head, only prints first ten lines of input.
@@ Line 61: / Line 61: @@
 As you can see fastq-mcf is able to deal with multiple files in the one command.
 = Reassessment of data quality =
@@ Line 66: / Line 69: @@
   fastqc --nogroup Read_1_q32l50.fastq.gz Read_2_q32l50.fastq.gz
-  firefox Read*q34l50*fastqc.html
+  firefox Read*q32l50*fastqc.html
 Look at the FastQC results and answer the following questions:
@@ Line 72: / Line 75: @@
 * What is the length of the reads?
 * Did qualities improve?

@@ Line 14: / Line 14: @@
 * FastQC: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/
 * FastqMcf: https://code.google.com/p/ea-utils/wiki/FastqMcf
-  module load ea-utils
+  module load FASTQC ea-utils
 * The data set you'll be using is downloaded from ENA (http://www.ebi.ac.uk/ena/data/view/SRP019027).
@@ Line 23: / Line 22: @@
 = View data set =
-  cd $HOME/i2rda_data/Quality_control_and_preprocessing
+  cd $HOME/i2rda_data/Quality_Control_and_Preprocessing
   zcat Read_1.fastq.gz |head
   zcat Read_2.fastq.gz |head
 = Assessment of data quality =
 Run FastQC on the raw data:
@@ Line 34: / Line 39: @@
 where:
-* --nogroup disables grouping of bases for reads >50bp. All reports will show data for every base in the read.
+* --nogroup, for visualisation purposes, prevents grouping of bases after read length of 50bp, so reports will show data for every base in the read.
 Look at the FastQC results and answer the following questions:
@@ Line 46: / Line 51: @@
 Trim reads using Fastq-Mcf:
-  fastq-mcf -o Read_1_q30l50.fastq -o Read_2_q30l50.fastq \
+  fastq-mcf -o Read_1_q32l50.fastq.qz -o Read_2_q32l50.fastq.qz -q 32 -l 50 \
- -q 30 -l 50 \
+  --qual-mean 32 adapters.fasta Read_1.fastq.gz Read_2.fastq.gz
 <ins>where</ins>:
@@ Line 55: / Line 59: @@
 * -l Minimum remaining sequence length
 * --qual-mean - Minimum mean quality score
 = Reassessment of data quality =
 Run FastQC on the trimmed reads:
-  fastqc --nogroup Read_1_q30l50.fastq Read_2_q30l50.fastq
+  fastqc --nogroup Read_1_q32l50.fastq.gz Read_2_q32l50.fastq.gz
-  firefox Read*q30l50*fastqc.html
+  firefox Read*q34l50*fastqc.html
 Look at the FastQC results and answer the following questions:

@@ Line 1: / Line 1: @@
 = Aims =
-NGS can be affected by a range of artefacts that arise during the library preparation and sequencing processes including low base
+You will use the following tools, which are available through the module load/unload system:
-quality, contamination with adapter sequences and biases in base composition, which can negatively impact the quality of the raw
-data for downstream analyses.
+* FastQC: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/
-In this part you will learn to:
+ module load FASTQC
-assess the intrinsic quality of raw reads using metrics generated by the sequencing platform (e.g. quality scores)
+* FastqMcf: https://code.google.com/p/ea-utils/wiki/FastqMcf
-pre-process data, i.e. trimming the poor quality bases and adapters from raw reads
+ module load ea-utils
-Type text like this in the terminal at the $ command prompt, then press the
+* The data set you'll be using is downloaded from ENA (http://www.ebi.ac.uk/ena/data/view/SRP019027).
-[Enter] key to run the command.
+* The reads belong to sample SRR769316, though this has been modified for course timing reasons.
 = View data set =
-  cd /home/training/Data/03_Quality_control_and_data_preprocessing
+  cd $HOME/i2rda_data/Quality_control_and_preprocessing
-  head Read_1.fastq
+  zcat Read_1.fastq.gz |head
-  head Read_2.fastq
+  zcat Read_2.fastq.gz |head
 = Assessment of data quality =
 Run FastQC on the raw data:
-  fastqc --nogroup Read_1.fastq Read_2.fastq
+  fastqc --nogroup Read_1.fastq.gz Read_2.fastq.gz
   firefox Read_*_fastqc.html &

← Older revision	Revision as of 13:00, 4 May 2017
(No difference)