今天上課老師讓我們按照PPT上操作順一遍DNA-seq流程，記錄如下（機(jī)器懂了人懵了：
##.fastq文件準(zhǔn)備
156? cp yeast_1.fastq /bios-analysis8/omics2022_post/cmd
? 157? cp yeast_2.fastq /bios-analysis8/omics2022_post/cmd
##若人多時(shí)，登入節(jié)點(diǎn)：
? 145? ssh c03n01
? 146? logout #退出節(jié)點(diǎn)
##運(yùn)行fastqc 查看原始數(shù)據(jù)的質(zhì)量
? 163? fastqc yeast_1.fastq
? 164? fastqc yeast_2.fastq
##運(yùn)行fastx_trimmer 去除reads末端質(zhì)量較差的部分 并查看質(zhì)量
? 166? fastx_trimmer -Q 33 -l 290 -i yeast_1.fastq -o yeast_trim_1.fastq
? 167? fastx_trimmer -Q 33 -l 200 -i yeast_2.fastq -o yeast_trim_2.fastq
###-l:last base to keep.default is entire read
? 168? fastqc yeast_trim_1.fastq
? 169? fastqc yeast_trim_2.fastq
##Mapping
###由于reads>100bps，所以使用bowtie2
? 171? bowtie2 -x /bios-store1/yeast_reference/Bowtie2Index_for_gtf/genome -1 yeast_trim_1.fastq -2 yeast_trim_2.fastq -S alignment.sam
Usage:
? bowtie2 [options]* -x <bt2-idx> {-1 <m1> -2 <m2> | -U <r> | --interleaved <i>} [-S <sam>]
? <bt2-idx>? Index filename prefix (minus trailing .X.bt2).
? ? ? ? ? ? NOTE: Bowtie 1 and Bowtie 2 indexes are not compatible.
? <m1>? ? ? Files with #1 mates, paired with files in <m2>.
? ? ? ? ? ? Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2).
? <m2>? ? ? Files with #2 mates, paired with files in <m1>.
? ? ? ? ? ? Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2).
? <r>? ? ? ? Files with unpaired reads.
? ? ? ? ? ? Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2).
? <i>? ? ? ? Files with interleaved paired-end FASTQ reads
? ? ? ? ? ? Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2).
? <sam>? ? ? File for SAM output (default: stdout)
? 173? less alignment.sam
? ##convert the sam file to bam
? 174? samtools view -Sb alignment.sam -o alignment.bam
##-Sb:input is SAM,output is BAM
? ##sorted the bam file
? 176? samtools sort alignment.bam align_sorted
? ##index the bam file
? 178 samtools index align_sorted.bam align_sorted.bam.bai
##可視化查看alignment result
? 201? samtools tview align_sorted.bam /bios-store1/yeast_reference/yeastgenome.fa
##查看alignment分析結(jié)果
? 204? /bios-store1/bin/bam_stat.py -i align_sorted.bam
##測(cè)序數(shù)據(jù)在基因組上的覆蓋度分析
? 207? /bios-store1/bin/bam2wig.py -s /bios-store1/yeast_reference/yeast_genome_size_chr.txt -u -q 20 -i align_sorted.bam -o align
Usage: bam2wig.py [options]
Convert BAM file into wig file. BAM file must be sorted and indexed using SAMtools.
Note: SAM format file is not supported.
-s:chromosome size file;-u:skip non-unique hit reads; -q:Minimum mapping quality to determine "uniquely mapped". default=30;
-o"output file(wig)
? 208? ls -lh
? 209? wc -l align.wig
? 210? more +1 align.wig
? 211? more +20000 align.wig
##genotyping calling analysis
? 212? /bios-store1/bin/samtools mpileup -D -q 20 -ugf /bios-store1/yeast_reference/yeastgenome.fa align_sorted.bam | bcftools view -vcg -D100 -> genotyping.vcf
? 213? less genotyping.vcf
##候選基因功能分析
###vcf文件轉(zhuǎn)為annovar input file
? 214? /bios-store1/program/annovar/convert2annovar.pl -format vcf4 ./genotyping.vcf? -outfile ./genotyping.inp
###候選基因注釋
? 218? /bios-store1/program/annovar/annotate_variation.pl -out ./genotyping -build AT? ./genotyping.inp? /bios-store1/program/annovar/atdb/
