===== Read preprocessing =====

1. Open a Terminal

2. Look at your fastq file

	cd /home/user/ngs-tutorial/1-quality_control_fastqc/1-raw_data/
	ll
	head solid.fastq

3. Generate reports for this fastq file
	fastqc solid.fastq -o /home/user/ngs-tutorial/1-quality_control_fastqc/2-fastqc_results/

4. Results have been saved in the results folder:
   ngs-tutorial > 1-quality_control_fastqc > 2-fastqc_results

These reads seem to need further processing...

5. Trim your sample based on its quality with a minimum quality threshold of 20:

	fastq_quality_trimmer -t 20 -i solid.fastq -o solid_t20.fastq

6. Remove the reads with less than a 90% bases with quality above 20:

	fastq_quality_filter -q 20 -p 90 -i solid.fastq -o solid_q20p90.fastq

7. Now we could rerun the quality report


===== Mapping =====

==== Create index ====

Index, create a index folder

	cd /home/user/ngs-tutorial/2-mapping/1-input_files/
	mkdir index
	bwa index -p index/hsapiens_chr20 Homo_sapiens.GRCh37.70.dna.chromosome.20.fa

==== Align ====

1. Paired-end mapping, 2 steps: first creating sai file, then the sam file

	bwa aln -t 2 -l 40 -k 2 -f ../2-results_bwa/test_1_pe.sai index/hsapiens_chr20 test_1.fq
	bwa aln -t 2 -l 40 -k 2 -f ../2-results_bwa/test_2_pe.sai index/hsapiens_chr20 test_2.fq
	
	bwa sampe -n 1 -f ../2-results_bwa/test_pe.sam index/hsapiens_chr20 ../2-results_bwa/test_1_pe.sai ../2-results_bwa/test_2_pe.sai test_1.fq test_2.fq


2. Creating BAM file with samtools and sort it

	cd /home/user/ngs-tutorial/2-mapping/2-results_bwa
	samtools view -S test_pe.sam -b -o test_pe.bam
	samtools sort test_pe.bam test_pe_sorted

==== Quality control of the alignment ====

	qualimap &

==== IGV visualitzation ====
	samtools index test_pe_sorted.bam
	igv &


===== Variant calling =====

An easy way to run variant calling

	cd /home/user/ngs-tutorial/3-variant_calling/
	./variant_calling

What we are doing is:

1. Remove duplicates

	java -jar MarkDuplicates.jar INPUT=aligned_withDup.bam OUTPUT=aligned.bam 

2. Count reads

	java -jar GenomeAnalysisTK.jar -T CountReads -R f000_reference.fa -I aligned.bam

3. Realignment

	java -jar GenomeAnalysisTK.jar -T RealignerTargetCreator -R f000_reference.fa -I aligned.bam -o f060_intervals2realign_aligned.list
	
	java -jar GenomeAnalysisTK.jar -T IndelRealigner -R f000_reference.fa -I f050_aligned.bam -targetIntervals f060_intervals2realign_aligned.list -o f070_realigned_aligned.bam

4. Variant calling

	java -jar GenomeAnalysisTK.jar -T UnifiedGenotyper -R f000_reference.fa -I f070_realigned_aligned.bam -glm SNP -o f080_snp_variants.vcf
	
	java -jar GenomeAnalysisTK.jar -T UnifiedGenotyper -R f000_reference.fa -I f070_realigned_aligned.bam -glm INDEL -o f080_indel_variants.vcf

5. Recalibration

	java -jar GenomeAnalysisTK.jar -T VariantFiltration -R f000_reference.fa -V f080_snp_variants.vcf --filterExpression "QD < 12.0" --filterName "LowConf" -o f090_snp_filtered.vcf