写在前面
转录组通常被用来寻找差异基因，但是有的时候我们想看一下已报道的基因是否存在可变剪切现象，即新转录本的挖掘，这个时候就需要利用StringTie+gffread来挖掘基因的新转录本。
StringTie
StringTie用于转录本的组装和转录组数据的量化。它使用一种新的网络流算法和可选的从头组装步骤来组装和量化代表每个基因位点的多个剪接变体的全长转录本。它的输入不仅可以包括其他转录汇编程序也可以使用的短读序列的比对，还可以包括从这些比对中组合的长序列的比对
1.安装.
二进制文件，安装过程请看官方说明。
下载链接：http://ccb.jhu.edu/software/stringtie/dl/stringtie-2.2.1.Linux_x86_64.tar.gz
##解压缩
tar xvf stringtie-2.2.1.Linux_x86_64.tar.gz
vim ~/.bashrc
source ~/.bashrc
2.数据预处理
输入文件是用TopHat或Hisat2比对后得到的bam文件。
3.基本参数
-p 线程数
-G 基因组注释文件
-o 输出的gtf文件
--merge 这是StringTie的一种特殊使用模式，与上面描述的程序集使用模式不同。在合并模式中，StringTie将GTF/GFF文件列表作为输入，并将这些转录合并/汇编为一组非冗余的转录。在新的差异分析流水线中使用这种模式，在多个RNA-Seq样本中生成一套统一的转录本(亚型)
4.正式运行
#批量输出注释文件
cat id | while read id; do stringtie -p 16 -G ref/Orysativa.gtf -o gtf/$id.gtf 01-align-ensembl/$id.bam ; done &
还有需要注意的是，如果输入的bam文件是来自于HISAT2比对的话，需要在HISAT2使用时加上--dta，这有利于stringtie的组装。
输出结果解读
Description of each column's values:
- seqname: Denotes the chromosome, contig, or scaffold for this transcript. Here the assembled transcript is on chromosome X.
- source: The source of the GTF file. Since this example was produced by StringTie, this column simply shows 'StringTie'.
- feature: Feature type; e.g., exon, transcript, mRNA, 5'UTR).
- start: Start position of the feature (exon, transcript, etc), using a 1-based index.
- end: End position of the feature, using a 1-based index.
- score: A confidence score for the assembled transcript. Currently this field is not used, and StringTie reports a constant value of 1000 if the transcript has a connection to a read alignment bundle.
- strand: If the transcript resides on the forward strand, '+'. If the transcript resides on the reverse strand, '-'.
- frame: Frame or phase of CDS features. StringTie does not use this field and simply records a ".".
- attributes
: A semicolon-separated list of tag-value pairs, providing additional information about each feature. Depending on whether an instance is a transcript or an exon and on whether the transcript matches the reference annotation file provided by the user, the content of the attributes field will differ. The following list describes the possible attributes shown in this column: * gene_id: A unique identifier for a single gene and its child transcript and exons based on the alignments' file name. * transcript_id: A unique identifier for a single transcript and its child exons based on the alignments' file name. * exon_number: A unique identifier for a single exon, starting from 1, within a given transcript. * reference_id: The transcript_id in the reference annotation (optional) that the instance matched. * ref_gene_id: The gene_id in the reference annotation (optional) that the instance matched. * ref_gene_name: The gene_name in the reference annotation (optional) that the instance matched. * cov: The average per-base coverage for the transcript or exon. * FPKM: Fragments per kilobase of transcript per million read pairs. This is the number of pairs of reads aligning to this feature, normalized by the total number of fragments sequenced (in millions) and the length of the transcript (in kilobases). * TPM: Transcripts per million. This is the number of transcripts from this particular gene normalized first by gene length, and then by sequencing depth (in millions) in the sample. A detailed explanation and a comparison of TPM and FPKM can be found [here](http://www.rna-seqblog.com/rpkm-fpkm-and-tpm-clearly-explained/), and TPM was defined [by B. Li and C. Dewey here](http://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-12-323).
#将多个转录本合并
stringtie --merge -G ref/Orysativa.gtf -e gtf/file1.gtf gtf/file2.gtf gtf/file3.gtf -o NIP0h.gtf
官网说还能将stringTie的count结果提取出来，用于Deseq2差异分析，这部分内容暂时用不到，往后用到的时候再来学习吧~
Gffcompare
当与参考注释(也作为GFF提供)进行比较时，gffcompare可用于比较、合并、注释和估计一个或多个GFF文件(“查询”文件)的准确性。
Assuming that Cufflinks' transcript assembly output file name is
cufflinks_asm.gtfand StringTie's output is instringtie_asm.gtf, while the reference annotation would be in a file called mm10.gff, the gffcompare commands would be
gffcompare -R -r mm10.gff -o cuffcmp cufflinks_asm.gtf</pre>
输出结果文件
cuffcmp.loci ：locus id的位置信息，个人理解为组装后的gene的local信息
cuffcmp.stats ：统计结果展示，看看就行了
cuffcmp.combined.gtf ：如果输入多个gtf组装文件的话，这个文件是将多个文件中的转录本并集输入到这个文件中
cuffcmp.stringtie_merged_new.gtf.refmap ：如果组装的gtf文件中的转录本either fully or partially match参考基因组转录本，则输入该文件中，也就是说组装后与参考基因组几乎完全匹配的转录本
cuffcmp.stringtie_merged_new.gtf.tmap :这个文件跟cuffcmp.stringtie_merged_new.gtf.refmap形式相似，但是输入的则是组装后与参考基因组较为近似的转录本
cuffcmp.tracking ：这个是比较重要的一个文件，里面含有cuffcompare软件对组装转录本分类定义的描述信息
2.1 cuffcmp.tracking文件
第1列 ：Cufflinks transfrag id，A unique internal id for the transfrag
第2列 ：Cufflinks locus id，A unique internal id for the locus
第3列 ：Reference gene id（如果没有则为"-"）
第4列 ：Reference transcript id（如果没有则为"-"）
第5列 ：class code，也就是组装转录本的分类信息
1 = Complete match of intron chain
2 c Contained
3 j Potentially novel isoform (fragment): at least one splice junction is shared with a reference transcript
4 e Single exon transfrag overlapping a reference exon and at least 10 bp of a reference intron, indicating a possible pre-mRNA fragment.
5 i A transfrag falling entirely within a reference intron
6 o Generic exonic overlap with a reference transcript
7 p Possible polymerase run-on fragment (within 2Kbases of a reference transcript)
8 r Repeat. Currently determined by looking at the soft-masked reference sequence and applied to transcripts where at least 50% of the bases are lower case
9 u Unknown, intergenic transcript
10 x Exonic overlap with reference on the opposite strand
11 s An intron of the transfrag overlaps a reference intron on the opposite strand (likely due to read mapping errors)
12 . (.tracking file only, indicates multiple classifications)</pre>
参考链接
1.StringTie https://ccb.jhu.edu/software/stringtie/
2.StringTie manual(附带输出结果解读) https://ccb.jhu.edu/software/stringtie/index.shtml?t=manual
3.gffcompare https://ccb.jhu.edu/software/stringtie/gffcompare.shtml
