转载1.Hisat2建立基因组索引：
First, using the python scripts included in the HISAT2 package, extract splice-site and exon information from the gene
annotation file:
$ extract_splice_sites.py gemome.gtf >genome.ss#得到剪接位点信息
$ extract_exons.py genome.gtf >genome.exon#得到外显子信息
Second, build a HISAT2 index:
$ hisat2-build --ss genome.ss --exon genome.exon genome.fa genome
备注extract_splice_sites.py 和 extract_exons.py 在hisat2软件包中涵盖了，这两步不是必须的，只是为了发现剪切位点，也可以直接：
$ hisat2-build genome.fa genome
2. 利用hisat2比对到基因组：
hisat2 -p 8 --dta -x genome -1 file1_1.fastq.gz -2 file1_2.fastq.gz -S file1.sam
hisat2 -p 8 --dta -x chrX_data/indexes/chrX_tran -1 file2_1.fastq.gz -2 file2_2.fastq.gz -S file2.sam
备注：--dta:输出转录组型的报告文件
-x：基因组索引
-S : 输出sam文件
-p: 线程数
其他参数：
Input:
-q query input files are FASTQ .fq/.fastq (default)
--qseq query input files are in Illumina's qseq format
-f query input files are (multi-)FASTA .fa/.mfa
-r query input files are raw one-sequence-per-line
-c , , are sequences themselves, not files
-s/--skip skip the first reads/pairs in the input (none)
-u/--upto stop after first reads/pairs (no limit)
-5/--trim5 trim bases from 5'/left end of reads (0)
-3/--trim3 trim bases from 3'/right end of reads (0)
--phred33 qualities are Phred+33 (default)
--phred64 qualities are Phred+64
--int-quals qualities encoded as space-delimited integers
Alignment:
-N max # mismatches in seed alignment; can be 0 or 1 (0)
-L length of seed substrings; must be >3, <32 (22)
-i interval between seed substrings w/r/t read len (S,1,1.15)
--n-ceil func for max # non-A/C/G/Ts permitted in aln (L,0,0.15)
--dpad include extra ref chars on sides of DP table (15)
--gbar disallow gaps within nucs of read extremes (4)
--ignore-quals treat all quality values as 30 on Phred scale (off)
--nofw do not align forward (original) version of read (off)
--norc do not align reverse-complement version of read (off)
Spliced Alignment:
--pen-cansplice penalty for a canonical splice site (0)
--pen-noncansplice penalty for a non-canonical splice site (12)
--pen-canintronlen penalty for long introns (G,-8,1) with canonical splice sites
--pen-noncanintronlen penalty for long introns (G,-8,1) with noncanonical splice sites
--min-intronlen minimum intron length (20)
--max-intronlen maximum intron length (500000)
--known-splicesite-infile provide a list of known splice sites
--novel-splicesite-outfile report a list of splice sites
--novel-splicesite-infile provide a list of novel splice sites
--no-temp-splicesite disable the use of splice sites found
--no-spliced-alignment disable spliced alignment
--rna-strandness Specify strand-specific information (unstranded)
--tmo Reports only those alignments within known transcriptome
--dta Reports alignments tailored for transcript assemblers
--dta-cufflinks Reports alignments tailored specifically for cufflinks
Scoring:
--ma match bonus (0 for --end-to-end, 2 for --local)
--mp , max and min penalties for mismatch; lower qual = lower penalty <2,6>
--sp , max and min penalties for soft-clipping; lower qual = lower penalty <1,2>
--np penalty for non-A/C/G/Ts in read/ref (1)
--rdg , read gap open, extend penalties (5,3)
--rfg , reference gap open, extend penalties (5,3)
--score-min min acceptable alignment score w/r/t read length
(L,0.0,-0.2)
Reporting:
(default) look for multiple alignments, report best, with MAPQ
OR
-k report up to alns per read; MAPQ not meaningful
OR
-a/--all report all alignments; very slow, MAPQ not meaningful
Effort:
-D give up extending after failed extends in a row (15)
-R for reads w/ repetitive seeds, try sets of seeds (2)
Paired-end:
--fr/--rf/--ff -1, -2 mates align fw/rev, rev/fw, fw/fw (--fr)
--no-mixed suppress unpaired alignments for paired reads
--no-discordant suppress discordant alignments for paired reads
Output:
-t/--time print wall-clock time taken by search phases
--un write unpaired reads that didn't align to
--al write unpaired reads that aligned at least once to
--un-conc write pairs that didn't align concordantly to
--al-conc write pairs that aligned concordantly at least once to
(Note: for --un, --al, --un-conc, or --al-conc, add '-gz' to the option name, e.g.
--un-gz , to gzip compress output, or add '-bz2' to bzip2 compress output.)
--quiet print nothing to stderr except serious errors
--met-file send metrics to file at (off)
--met-stderr send metrics to stderr (off)
--met report internal counters & metrics every secs (1)
--no-head supppress header lines, i.e. lines starting with @
--no-sq supppress @SQ header lines
--rg-id set read group id, reflected in @RG line and RG:Z: opt field
--rg add ("lab:value") to @RG line of SAM header.
Note: @RG line only printed when --rg-id is set.
--omit-sec-seq put '*' in SEQ and QUAL fields for secondary alignments.
Performance:
-o/--offrate override offrate of index; must be >= index's offrate
-p/--threads number of alignment threads to launch (1)
--reorder force SAM output order to match order of input reads
--mm use memory-mapped I/O for index; many 'bowtie's can share
Other:
--qc-filter filter out reads that are bad according to QSEQ filter
--seed seed for random number generator (0)
--non-deterministic seed rand. gen. arbitrarily instead of using read attributes
--version print version information and quit
-h/--help print this usage message
3. 将sam文件sort并转化成bam:
$ samtools sort -@ 8 -o file1.bam file1.sam
$ samtools sort -@ 8 -o file2.bam file2.sam
4. 组装转录本：
$ stringtie -p 8 -G genome.gtf -o file1.gtf –l file1 file1.bam
$ stringtie -p 8 -G genome.gtf -o file2.gtf –l file2 file2.bam
lncRNA (-f 0.01 -a 10 -j 1 -c 0.01)
其中：
-G reference annotation to use for guiding the assembly process (GTF/GFF3)
-l name prefix for output transcripts (default: STRG)
-f minimum isoform fraction (default: 0.1)
-m minimum assembled transcript length (default: 200)
-o output path/file name for the assembled transcripts GTF (default: stdout)
-a minimum anchor length for junctions (default: 10)
-j minimum junction coverage (default: 1)
-t disable trimming of predicted transcripts based on coverage
(default: coverage trimming is enabled)
-c minimum reads per bp coverage to consider for transcript assembly
(default: 2.5)
-v verbose (log bundle processing details)
-g gap between read mappings triggering a new bundle (default: 50)
-C output a file with reference transcripts that are covered by reads
-M fraction of bundle allowed to be covered by multi-hit reads (default:0.95)
-p number of threads (CPUs) to use (default: 1)
-A gene abundance estimation output file
-B enable output of Ballgown table files which will be created in the
same directory as the output GTF (requires -G, -o recommended)
-b enable output of Ballgown table files but these files will be
created under the directory path given as
-e only estimate the abundance of given reference transcripts (requires -G)
-x do not assemble any transcripts on the given reference sequence(s)
-h print this usage message and exit
5. 合并所有样本的gtf文件
$ stringtie --merge -p 8 -G genome.gtf -o stringtie_merged.gtf mergelist.txt
6. 新转录本的注释（lncRNA必备，普通转录组忽略）
gffcompare –r genomegtf –G –o merged stringtie_merged.gtf
备注：gffcompare 是独立软件，下载地址http://ccb.jhu.edu/software/stringtie/gff.shtml，结果如下;
= Predicted transcript has exactly the same introns as the reference transcript
c Predicted transcript is contained within the reference transcript
j Predicted transcript is a potential novel isoform that shares at least one splice junction with a reference transcript
e Predicted single-exon transcript overlaps a reference exon plus at least 10 bp of a reference intron, indicating a possible pre-mRNA fragment
i Predicted transcript falls entirely within a reference intron
o Exon of predicted transcript overlaps a reference transcript
p Predicted transcript lies within 2 kb of a reference transcript (possible polymerase run-on fragment)
r Predicted transcript has >50% of its bases overlapping a soft-masked (repetitive) reference sequence
u Predicted transcript is intergenic in comparison with known reference transcripts
x Exon of predicted transcript overlaps reference but lies on the opposite strand
s Intron of predicted transcript overlaps a reference intron on the opposite strand
7. 转录本定量和下游ballgown软件原始文件构建：
$ stringtie –e –B -p 8 -G stringtie_merged.gtf -o ballgown/file1/file1.gtf file1.bam
$ stringtie –e –B -p 8 -G stringtie_merged.gtf -o ballgown/file2/file2.gtf file2.bam
8. Ballgown差异表达分析：
>library(ballgown)
>library(RSkittleBrewer)
>library(genefilter)
>library(dplyr)
>library(devtools)
>pheno_data = read.csv("geuvadis_phenodata.csv")#读取表型数据
>bg_chrX = ballgown(dataDir = "ballgown", samplePattern = "file", pData=pheno_data)#读取表达量
>bg_chrX_filt = subset(bg_chrX,"rowVars(texpr(bg_chrX)) >1",genomesubset=TRUE)#过滤低表达量基因
>results_transcripts = stattest(bg_chrX_filt,feature="transcript",covariate="sex",adjustvars =c("population"), getFC=TRUE, meas="FPKM")#差异表达分析，运用的是一般线性模型，比较组sex，影响因素：population
>results_genes = stattest(bg_chrX_filt, feature="gene",covariate="sex", adjustvars = c("population"), getFC=TRUE,meas="FPKM")#基因差异表达
>results_transcripts=data.frame(geneNames=ballgown::geneNames(bg_chrX_filt),geneIDs=ballgown::geneIDs(bg_chrX_filt), results_transcripts)#增加基因名字，id
>results_transcripts = arrange(results_transcripts,pval)#按pval sort
>results_genes = arrange(results_genes,pval)
>write.csv(results_transcripts, "chrX_transcript_results.csv",
row.names=FALSE)
>write.csv(results_genes, "chrX_gene_results.csv",
row.names=FALSE)
>subset(results_transcripts,results_transcripts$qval<0.05)
>subset(results_genes,results_genes$qval<0.05)
9. 结果可视化：
>tropical= c('darkorange', 'dodgerblue',
'hotpink', 'limegreen', 'yellow')
>palette(tropical)
>fpkm = texpr(bg_chrX,meas="FPKM")
>fpkm = log2(fpkm+1)
>boxplot(fpkm,col=as.numeric(pheno_data$sex),las=2,ylab='log2(FPKM+1)')
>ballgown::transcriptNames(bg_chrX)[12]
## 12
## "NM_012227"
>ballgown::geneNames(bg_chrX)[12]
## 12
## "GTPBP6"
>plot(fpkm[12,] ~ pheno_data$sex, border=c(1,2),
main=paste(ballgown::geneNames(bg_chrX)[12],' : ',
ballgown::transcriptNames(bg_chrX)[12]),pch=19, xlab="Sex",
ylab='log2(FPKM+1)')
>points(fpkm[12,] ~ jitter(as.numeric(pheno_data$sex)),
col=as.numeric(pheno_data$sex))
>plotTranscripts(ballgown::geneIDs(bg_chrX)[1729], bg_chrX, main=c('Gene XIST in sample ERR188234'), sample=c('ERR188234'))
>plotMeans('MSTRG.56', bg_chrX_filt,groupvar="sex",legend=FALSE)