Cufflinks的使用詳解之--cuffdiff

Cufflinks下主要包含cufflinks,cuffmerge,cuffcompare和cuffdiff等幾支主要的程序。主要用于轉(zhuǎn)錄組的組裝和差異表達分析。

The Cufflinks RNA-Seq workflow

一怀吻、Cuffdiff簡介

用于尋找轉(zhuǎn)錄子表達的顯著性差異蝉仇。

二、Cuffdiff使用方法

（默認Linux操作洼裤，此處省略安裝步驟）

cuffdiff主要是發(fā)現(xiàn)轉(zhuǎn)錄本表達，剪接溪王，啟動子使用的明顯變化腮鞍。

Usage:   cuffdiff [options] <transcripts.gtf> <sample1_hits.sam> <sample2_hits.sam> [... sampleN_hits.sam]
   Supply replicate SAMs as comma separated lists for each condition: sample1_rep1.sam,sample1_rep2.sam,...sample1_repM.sam

其中transcripts.gtf是由cufflinks，cuffcompare莹菱，cuffmerge所生成的文件移国，或是由其它程序生成的。一個樣本有多個replicate道伟，用逗號隔開迹缀。sample多于一個時使碾，cuffdiff將比較samples間的基因表達的差異性。

cuffdiff接受bam/sam或cuffquant的CXB文件祝懂，同時也可以接受bam與sam的混合文件票摇，不能接受bam/sam和CXB的混合文件。

三砚蓬、Cuffdiff參數(shù)說明

General Options:
  -o/--output-dir              write all output files to this directory              [ default:     ./ ]    #輸出的文件夾目錄
  -L/--labels                  comma-separated list of condition labels    #給每個sample一個樣品名或者一個環(huán)境條件一個lable
  --FDR                        False discovery rate used in testing                  [ default:   0.05 ]    #允許的false discovery rate
  -M/--mask-file               ignore all alignment within transcripts in this file  [ default:   NULL ]    #提供GFF文件矢门。Cufflinks將忽略比對到該GTF文件的transcripts中的reads。該文件中常常是rRNA的注釋灰蛙，也可以包含線立體和其它希望忽略的transcripts的注釋祟剔。將這些不需要的RNA去除后，對計算mRNA的表達量是有利的摩梧。
  -C/--contrast-file           Perform the constrasts specified in this file         [ default:   NULL ]    #比對指定文件
  -b/--frag-bias-correct       use bias correction - reference fasta required        [ default:   NULL ]    #提供一個fasta文件來指導Cufflinks運行新的bias detection and correction algorithm物延。這樣能明顯提高轉(zhuǎn)錄子豐度計算的精確性。
  -u/--multi-read-correct      use 'rescue method' for multi-reads                   [ default:  FALSE ]    #讓Cufflinks來做initial estimation步驟仅父，從而更精確衡量比對到genome多個位點的reads教届。
  -p/--num-threads             number of threads used during quantification          [ default:      1 ]    #使用的CPU線程數(shù)
  --no-diff                    Don't generate differential analysis files            [ default:  FALSE ]    #不需要生成差異分析文件
  --no-js-tests                Don't perform isoform switching tests                 [ default:  FALSE ]    #不需要進行isoform轉(zhuǎn)換測試
  -T/--time-series             treat samples as a time-series                        [ default:  FALSE ]    #讓Cuffdiff來按樣品順序來比對樣品，而不是對所有的samples都進行兩兩比對驾霜。即第二個SAM和第一個SAM比案训；第三個SAM和第二個SAM比；第四個SAM和第三個SAM比...
  --library-type               Library prep used for input reads                     [ default:  below ]    #處理的reads具有鏈特異性粪糙。比對結(jié)果中將會有個XS標簽强霎。一般Illumina數(shù)據(jù)的library-type為 fr-unstranded。
  --dispersion-method          Method used to estimate dispersion models             [ default:  below ]    #用于估計分散模型的方法
  --library-norm-method        Method used to normalize library sizes                [ default:  below ]    #用于標準化庫大小的方法

四蓉冈、Cuffdiff輸出

1. FPKM tracking files

cuffdiff計算每個樣本中的轉(zhuǎn)錄本城舞，初始轉(zhuǎn)錄本和基因的FPKM。其中寞酿，基因和初始轉(zhuǎn)錄本的FPKM的計算是在每個轉(zhuǎn)錄本group和基因group中的轉(zhuǎn)錄本的FPKM的求和家夺。

1|isoforms.fpkm_tracking             Transcript FPKMs
2|genes.fpkm_tracking                Gene FPKMs. Tracks the summed FPKM of transcripts sharing each gene_id
3|cds.fpkm_tracking                  Coding sequence FPKMs. Tracks the summed FPKM of transcripts sharing each p_id, independent of tss_id
4|tss_groups.fpkm_tracking           Primary transcript FPKMs. Tracks the summed FPKM of transcripts sharing each tss_id

文件格式：

1|tracking_id class_code  nearest_ref_id  gene_id gene_short_name tss_id  locus   length  coverage    P1_FPKM P1_conf_lo  P1_conf_hi  P1_status   P2_FPKM P2_conf_lo  P2_conf_hi  P2_status   P3_FPKM P3_conf_lo  P3_conf_hi  P3_status
2|ENST00000000233 -   -   ENSG00000004059 ARF5    -   7:127220671-127242198   1103    -   58.3768 0   139.888 OK  47.3478 0   113.046 OK  78.9705 0   184.419 OK

注：P1、P2伐弹、P3為樣本名

2. Count tracking files

評估每個樣本中來自每個 transcript, primary transcript, and gene的fragment數(shù)目拉馋。其中primary transcript, and gene的fragment數(shù)目是每個primary transcript group或gene group中trancript的數(shù)目之和。

1|isoforms.count_tracking             Transcript counts
2|genes.count_tracking                Gene counts. Tracks the summed counts of transcripts sharing each gene_id
3|cds.count_tracking                  Coding sequence counts. Tracks the summed counts of transcripts sharing each p_id, independent of tss_id
4|tss_groups.count_tracking           Primary transcript counts. Tracks the summed counts of transcripts sharing each tss_id

文件格式：

1|tracking_id P1_count    P1_count_variance   P1_count_uncertainty_var    P1_count_dispersion_var P1_status   P2_count    P2_count_variance   P2_count_uncertainty_var    P2_count_dispersion_var P2_status   P3_count    P3_count_variance   P3_count_uncertainty_var    P3_count_dispersion_var P3_status
2|ENST00000000233 1226.79 733396  0   591186  OK  992.56  474498  0   376391  OK  1661.82 1.22994e+06 0   1.22994e+06 OK

3. Read group tracking files

計算在每個repulate中每個transcript惨好， primary transcript和gene的表達量和frage數(shù)目煌茴。

1|isoforms.read_group_tracking      Transcript read group tracking
2|genes.read_group_tracking         Gene read group tracking. Tracks the summed expression and counts of transcripts sharing each gene_id in each replicate
3|cds.read_group_tracking           Coding sequence FPKMs. Tracks the summed expression and counts of transcripts sharing each p_id, independent of tss_id in each replicate
4|tss_groups.read_group_tracking    Primary transcript FPKMs. Tracks the summed expression and counts of transcripts sharing each tss_id in each replicate

文件格式：

1|tracking_id condition   replicate   raw_frags   internal_scaled_frags   external_scaled_frags   FPKM    effective_length    status   
2|ENST00000000233 MofRCC  1   1307.38 1182.81 1182.81 56.4898 -   OK

4. Differential expression test files

對于splicing transcript，primary transcripts, genes, and coding sequences.樣本之間的表達差異檢驗日川。對于每一對樣本x和y蔓腐，都會有以下四個文件：

1|isoform_exp.diff                  Transcript differential FPKM.
2|gene_exp.diff                     Gene differential FPKM. Tests difference sin the summed FPKM of transcripts sharing each gene_id
3|tss_group_exp.diff                Primary transcript differential FPKM. Tests differences in the summed FPKM of transcripts sharing each tss_id
4|cds_exp.diff                      Coding sequence differential FPKM. Tests differences in the summed FPKM of transcripts sharing each p_id independent of tss_id

文件格式：

1|test_id gene_id gene    locus   sample_1    sample_2    status  value_1 value_2 log2(fold_change)   test_stat   p_value q_value significant
2|ENST00000000233 ENSG00000004059 ARF5    7:127220671-127242198   MofRCC  NofRCC  OK  58.3768 47.3478 -0.302097   -0.212748   0.7584  0.992833    no

5. Differential splicing tests – splicing.diff

對于每個primary transcript，鑒定的不同的isoform的差異性龄句。只有2個或2個以上的isoforms的primary transcript存在回论。
文件格式：

1|test_id gene_id gene    locus   sample_1    sample_2    status  value_1 value_2 sqrt(JS)    test_stat   p_value q_value significant

6. Differential coding output – cds.diff

對于每個基因散罕，它的cds的鑒定。樣本間的輸出cds的差異性傀蓉。只有2個或2個以上的cds（multi-protein genes）列舉在文件中笨使。
文件格式：

1|test_id gene_id gene    locus   sample_1    sample_2    status  value_1 value_2 sqrt(JS)    test_stat   p_value q_value significant

7. Differential promoter use – promoters.diff

樣本間啟動子使用的差異性。只有表達2個或2個以上isoform的基因列舉在這里僚害。
文件格式：

1|test_id gene_id gene    locus   sample_1    sample_2    status  value_1 value_2 sqrt(JS)    test_stat   p_value q_value significant

8. Read group info – read_groups.info

每個repulate硫椰，在進行定量分析時，cuffdiff的關(guān)鍵屬性會列出萨蚕。
文件格式：

1|file    condition   replicate_num   total_mass  norm_mass   internal_scale  external_scale
2|/PROJ/*/Quantification/P1/abundances.cxb    MofRCC  0   2.8904e+07  2.44127e+07 1.20839 1

9. Run info – run.info

運行信息靶草。