一、測序數(shù)據(jù)的準備

新建工作目錄，GATK-analysis

mkdir RNASeq-analysis
cd RNASeq-analysis/

新建data文件夾；
mkdir data

1.1 公司返回的測序下機數(shù)據(jù)

例如：6個NGS測序樣本；
Raw data: Sample1_R1.fq.gz; Sample1_R2.fq.gz; Sample2_R1.fq.gz; Sample2_R2.fq.gz; Sample3_R1.fq.gz; Sample3_R2.fq.gz; Sample4_R1.fq.gz; Sample4_R2.fq.gz; Sample5_R1.fq.gz; Sample5_R2.fq.gz; Sample6_R1.fq.gz; Sample6_R2.fq.gz;

1.2 從SRA數(shù)據(jù)庫下載數(shù)據(jù)

第一步，獲取SRA編號Accession: PRJNA******/SRP******；

第二步，下載安裝sra-tookit

conda install sra-tools

第三步，下載數(shù)據(jù)

prefetch SRR******

第四步，對數(shù)據(jù)進行拆分

使用 SRA-toolkit 軟件包里的 fastq-dump 對數(shù)據(jù)進行拆分，并轉(zhuǎn)換為 fastq 格式。
nohup fastq-dump --split-3 SRR****** 1>SRR******.log 2>SRR******.err &
將測序數(shù)據(jù)全部置于data文件夾，
mv Sample* data/

二、對數(shù)據(jù)進行質(zhì)控和過濾

for file in `ls *_R1.fq.gz | perl -lpe 's/_R1.fq.gz//'`; do fastp -i ${file}_R1.fq.gz -I ${file}_R2.fq.gz -o ${file}_R1.clean.fq.gz -O ${file}_R2.clean.fq.gz; done

三、參考基因組準備

主要準備基因組文件Species.genome.fasta;
新建references文件夾：
mkdir references
將參考文件置于references文件夾；
mv Species.genome.fasta ~/GATK-analysis/references/

四、bwa比對

新建bwa工作目錄：

 mkdir bwa
cd bwa '
vim bwa.sh 

#bwa.sh
bwa index ../references/Species.genome.fasta
for file in `ls ../data/*_R1.fq.gz | perl -lpe 's/_R1.fq.gz//'`; do 
    bwa mem -t 4 -R '@RG\tID:foo\tPL:illumina\tSM:${file}' ../references/Species.genome.fasta ${file}_R1.fq.gz ${file}_R2.fq.gz > ${file}.sam
    samtools view -b ${file}.sam > ${file}.bam
    samtools sort ${file}.bam > ${file}.sorted.bam
    samtools index ${file}.sorted.bam
done

運行bwa:
nohup bash bwa.sh &

五、GATK calling SNP

直接下載安裝：
wget https://github.com/broadinstitute/gatk/releases/download/4.1.7.0/gatk-4.1.7.0.zip
欲了解詳情，參見官方文檔：
GATK 4.1.7
新建工作目錄：
mkdir gatk
cd gatk
將bwa生成的排序文件移至gatk目錄，并建立參考基因組的軟連接：

mv ../bwa/*.sorted.bam .
ln -s ../references/Species.genome.fasta .

編寫gatk腳本：
vim gatk.sh

samtools faidx Species.genome.fasta
gatk CreateSequenceDictionary -R Species.genome.fasta -O Species.genome.dict
for file in *.sorted.bam;do
    gatk MarkDuplicates -I ${file} -O ${file}.markdup.bam -M ${file}.markdup_metrics.txt
    samtools index ${file}.markdup.bam
    gatk HaplotypeCaller -R Species.genome.fasta --emit-ref-confidence GVCF -I ${file}.markdup.bam -O ${file}.g.vcf
    gatk GenotypeGVCFs -R Species.genome.fasta -V ${file}.g.vcf -O ${file}.vcf    
    bgzip -f ${file}.vcf
    tabix -p vcf ${file}.vcf.gz
    gatk SelectVariants -select-type SNP -V ${file}.vcf.gz -O ${file}.snp.vcf.gz    
    gatk VariantFiltration -V ${file}.snp.vcf.gz --filter-expression "QD < 2.0 || MQ < 40.0 || FS > 60.0 || SOR > 3.0 || MQRankSum < -12.5 || ReadPosRankSum < -8.0" --filter-name "PASS" -O ${file}.snp.filter.vcf.gz
    gatk SelectVariants -select-type INDEL -V ${file}.vcf.gz -O ${file}.indel.vcf.gz
    gatk VariantFiltration -V ${file}.indel.vcf.gz --filter-expression "QD < 2.0 || FS > 200.0 || SOR > 10.0 || MQRankSum < -12.5 || ReadPosRankSum < -8.0" --filter-name "PASS" -O ${file}.indel.filter.vcf.gz
    gatk MergeVcfs -I ${file}.snp.filter.vcf.gz -I ${file}.indel.filter.vcf.gz -O ${file}.filter.vcf.gz
done

運行GATK：
nohup bash gatk.sh &

得到結(jié)果文件${file}.filter.vcf.gz，里面包含SNP和indel。

參考：

1. Shifu Chen, Yanqing Zhou, Yaru Chen, Jia Gu; fastp: an ultra-fast all-in-one FASTQ preprocessor, Bioinformatics, Volume 34, Issue 17, 1 September 2018, Pages i884–i890, https://doi.org/10.1093/bioinformatics/bty560
2. Li H., Handsaker B., Wysoker A., Fennell T., Ruan J., Homer N., Marth G., Abecasis G., Durbin R. and 1000 Genome Project Data Processing Subgroup (2009) The Sequence alignment/map (SAM) format and SAMtools. Bioinformatics, 25, 2078-9. [PMID: 19505943]
3. Li H A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics. 2011 Nov 1;27(21):2987-93. Epub 2011 Sep 8. [PMID: 21903627]
4. Genome Analysis Toolkit