生物学重复：指样本重复，比如3只小鼠，同时做一种处理，就是三个生物学重复。
技术重复：一般是三次实验，比如对一块组织，提了三次RNA，做三次real time。
##HISAT Indexs
genome: HFM index for reference
genome_snp: Hierarchical Graph FM index (HGFM) index for reference plus SNPs
genome_tran: HGFM index for reference plus transcripts
genome_snp_tran: HGFM index for reference plus SNPs and transcripts
1. 统计 reads length
1) 统计fastq reads length
awk 'NR%4 == 2 {lengths[length($0)]++ ; counter++} END {for (l in lengths) {print l, lengths[l]}; print "total reads: " counter}' file.fastq
2) 统计bam中mapped reads length
samtools view ${id%%.*}_mapped.bam|awk 'BEGIN{OFS="\t"}{a[length($10)]++}END{for(i in a) print i,a[i]}' >${id%%.*}_mapped_read_length.txt
2. samtools view
提取比对到参考序列上的比对结果
$ samtools view -bF 4 abc.bam > abc.F.bam
提取paired reads中两条reads都比对到参考序列上的比对结果，只需要把两个4+8的值12作为过滤参数即可
$ samtools view -bF 12 abc.bam > abc.F12.bam
提取没有比对到参考序列上的比对结果
$ samtools view -bf 4 abc.bam > abc.f.bam
提取bam文件中比对到caffold1上的比对结果，并保存到sam文件格式
$ samtools view abc.bam scaffold1 > scaffold1.sam
提取scaffold1上能比对到30k到100k区域的比对结果
$ samtools view abc.bam scaffold1:30000-100000 $gt; scaffold1_30k-100k.sam
根据fasta文件，将 header 加入到 sam 或 bam 文件中
$ samtools view -T genome.fasta -h scaffold1.sam > scaffold1.h.sam
To get theunmappedreads from a bam file use :
samtools view -f 4 file.bam > unmapped.sam, the output will be in sam
to get the output in bam use : samtools view -b -f 4 file.bam > unmapped.bam
To get only the mapped reads use the parameter 'F', which works like-v of grep and skips the alignments for a specific flag.
samtools view -b -F 4 file.bam > mapped.bam
From themanual; there are different int codes you can use with the parameter 'f', based on what you want :
-f INT Only output alignments with all bits in INT present in
the FLAG field. INT can be in hex in the format of /^0x[0-9A-F]+/ [0]
Each bit in the FLAG field is defined as:
Flag Chr Description
0x0001 p the read is paired in sequencing
0x0002 P the read is mapped in a proper pair
0x0004 u the query sequence itself is unmapped
0x0008 U the mate is unmapped
0x0010 r strand of the query (1 for reverse)
0x0020 R strand of the mate
0x0040 1 the read is the first read in a pair
0x0080 2 the read is the second read in a pair
0x0100 s the alignment is not primary
0x0200 f the read fails platform/vendor quality checks
0x0400 d the read is either a PCR or an optical duplicate
Like for getting theuniquereads (a single read mapping at one best position); I use :
-q INT Skip alignments with MAPQ smaller than INT [0]
samtools view -bq 1 file.bam > unique.bam