全长转录组 | 三代全长转录组分析流程（PacBio & ONT ）-- Flair

#创建Flair软件环境，并安装
$ conda create -n flair -c conda-forge -c bioconda flair
$ conda activate flair
$ flair [align/correct/...]

$ docker pull brookslab/flair:latest
$ docker run -w /usr/data -v [your_path_to_data]:/usr/data brookslab/flair:latest flair [align/correct/...]

$ flair align -g genome.fa -r <reads.fq>|<reads.fa> [options]

--reads     Raw reads in fasta or fastq format. This argument accepts multiple
            (comma/space separated) files. # 下机测序序列，接受.fasta 或 .fastq格式文件；多个文件可以逗号/空格分开。

At least one of the following arguments is required （至少提供一个）:
--genome    Reference genome in fasta format. Flair will minimap index this file
            unless there already is a .mmi file in the same location. # 基因组参考序列(.fa)，minimap会自动建立索引。
--mm_index  If there already is a .mmi index for the genome it can be supplied
            directly using this option. # 可以直接输入minimap索引文件 .mmi。

--help              Show all options. #帮助命令。
--output            Name base for output files (default: flair.aligned). You can supply
                    an output directory (e.g. output/flair_aligned) but it has to exist;
                    Flair will not create it. If you run the same command twice, Flair
                    will overwrite the files without warning. #输出文件夹路径和文件前缀，文件夹必须自己创建。
--threads           Number of processors to use (default 4). #线程，默认为4。
--junction_bed      Annotated isoforms/junctions bed file for splice site-guided
                    minimap2 genomic alignment. #异构体/连接位点注释的bed文件，用于辅助minimap基因组比对。
--nvrna             Use native-RNA specific alignment parameters for minimap2 (-u f -k 14) #RNA直接测序选项。
--quality           Minimum MAPQ score of read alignment to the genome. The default is 1,
                    which is the lowest possible score. #比对的MAPQ值，默认为最低的1。
-N                  Retain at most INT secondary alignments from minimap2 (default 0). Please
                    proceed with caution, changing this setting is only useful if you know
                    there are closely related homologs elsewhere in the genome. It will
                    likely decrease the quality of Flair's final results. #保留几个次好的比对结果，默认为0。
--quiet             Dont print progress statements. #不输出过程。

usage: flair correct -q query.bed12 [-f annotation.gtf]|[-j introns.tab] -g genome.fa [options]

--query             Uncorrected bed12 file, e.g. output of flair align. #上一步比对后的bed12文件。
--genome            Reference genome in fasta format. #基因组参考文件。

At least one of the following arguments is required:
--shortread         Bed format splice junctions from short-read sequencing. You can
                    generate these from SAM format files using the junctions_from_sam
                    program that comes with Flair. # 来自于二代短读长测序的剪切位点（bed文件格式），可以利用FLAIR自带的junctions_from_sam脚本，将比对后生成的SAM文件转化为BED文件。
--gtf               GTF annotation file. #基因组注释文件。

--help              Show all options #帮助命令。
--output            Name base for output files (default: flair). You can supply an
                    output directory (e.g. output/flair) but it has to exist; Flair
                    will not create it. If you run the same command twice, Flair will
                    overwrite the files without warning.  #输出文件夹路径和文件前缀，文件夹必须自己创建。
--threads           Number of processors to use (default 4). #线程，默认为4。
--nvrna             Specify this flag to make the strand of a read consistent with
                    the input annotation during correction. #RNA直接测序选项。
--ss_window         Window size for correcting splice sites (default 15). #校正剪切位点的‘窗口(范围)’大小，默认值为15。
--print_check       Print err.txt with step checking. #输出报错信息。

usage: flair collapse -g genome.fa -q <query.bed> -r <reads.fq>/<reads.fa> [options]

--query     Bed file of aligned/corrected reads #比对完成/经过校正的序列
--genome    FastA of reference genome #参考基因组
--reads     FastA/FastQ files of raw reads, can specify multiple files #原始三代测序数据fasta/fastq， 可以指定多个。

--help              Show all options. #帮助
--output            Name base for output files (default: flair.collapse). #命名输出文件，默认为flair.collapse。
                    You can supply an output directory (e.g. output/flair_collapse) #指定输出文件夹。
--threads           Number of processors to use (default: 4). #线程数，默认为4。
--gtf               GTF annotation file, used for renaming FLAIR isoforms to
                    annotated isoforms and adjusting TSS/TESs. #gtf注释文件，用于FLAIR聚类的isoform的重新命名，调整转录本起始和结束位点。
--generate_map      Specify this argument to generate a txt file of read-isoform
                    assignments (default: not specified).#生成序列对应isoform的文本文件，默认不指定。
--annotation_reliant        Specify transcript fasta that corresponds to transcripts
                    in the gtf to run annotation-reliant flair collapse; to ask flair
                    to make transcript sequences given the gtf and genome fa, use
                    --annotation_reliant generate. #产生相应的转录本fasta序列文件。

--support           Minimum number of supporting reads for an isoform; if s < 1,
                    it will be treated as a percentage of expression of the gene
                    (default: 3). #最小几个序列支持一个isoform，默认为3。
--stringent         Specify if all supporting reads need to be full-length (80%
                    coverage and spanning 25 bp of the first and last exons). #支持序列必须都是全长（80%的覆盖率，第一个和最后一个外显子至少有25个碱基）
--check_splice      Enforce coverage of 4 out of 6 bp around each splice site and
                    no insertions greater than 3 bp at the splice site. Please note:
                    If you want to use --annotation_reliant as well, set it to
                    generate instead of providing an input transcripts fasta file,
                    otherwise flair may fail to match the transcript IDs.
                    Alternatively you can create a correctly formatted transcript
                    fasta file using gtf_to_psl #至少覆盖剪切位点6个中的4个，插入序列不能大约3bp。
--trust_ends        Specify if reads are generated from a long read method with
                    minimal fragmentation. #如果序列来自长序列建库方法（最小程度的打断）
--quality           Minimum MAPQ of read assignment to an isoform (default: 1). #序列归为isoform最低的MAPQ值。

--longshot_bam      BAM file from Longshot containing haplotype information for each read. #包含单倍型信息的BAM文件。
--longshot_vcf      VCF file from Longshot. #包含变异信息的VCF文件。

--end_window        Window size for comparing transcripts starts (TSS) and ends
                    (TES) (default: 100). #比较转录本起始和终止的窗口大小，默认为100。
--promoters         Promoter regions bed file to identify full-length reads. #启动子区域的bed文件以鉴定全长序列。
--3prime_regions    TES regions bed file to identify full-length reads. #转录本终止区域的bed文件以鉴定全长序列。
--no_redundant      <none,longest,best_only> (default: none). For each unique
                    splice junction chain, report options include:
                            - none          best TSSs/TESs chosen for each unique
                                            set of splice junctions #对每一个剪切位点选择最好的起始和终止。
                            - longest       single TSS/TES chosen to maximize length #选择最长的。
                            - best_only     single most supported TSS/TES #单个支持最多的。
--isoformtss        When specified, TSS/TES for each isoform will be determined
                    from supporting reads for individual isoforms (default: not
                    specified, determined at the gene level). #每一个isoform的转录起始和终止由支持它的序列所决定。
--no_gtf_end_adjustment     Do not use TSS/TES from the input gtf to adjust
                    isoform TSSs/TESs. Instead, each isoform will be determined
                    from supporting reads. #不使用注释文件去校正isoform的起始和终止。
--max_ends          Maximum number of TSS/TES picked per isoform (default: 2). #每一个isoform选中的TSS/TES的最大数值，默认为2。
--filter            Report options include:
                            - nosubset      any isoforms that are a proper set of
                                            another isoform are removed #同时归为其它类的isoform被去除。
                            - default       subset isoforms are removed based on support #基于支持数值去除isoforms子集。
                            - comprehensive default set + all subset isoforms
                            - ginormous     comprehensive set + single exon subset
                                            isoforms

--temp_dir          Directory for temporary files. use "./" to indicate current
                    directory (default: python tempfile directory). #指定临时文件夹。
--keep_intermediate         Specify if intermediate and temporary files are to
                    be kept for debugging. Intermediate files include:
                    promoter-supported reads file, read assignments to
                    firstpass isoforms. #保留中间文件。
--fusion_dist       Minimium distance between separate read alignments on the
                    same chromosome to be considered a fusion, otherwise no reads
                    will be assumed to be fusions. #融合基因在同一个染色体上的分开距离。
--mm2_args          Additional minimap2 arguments when aligning reads first-pass
                    transcripts; separate args by commas, e.g. --mm2_args=-I8g,--MD.
--quiet             Suppress progress statements from being printed. #过程不输出。
--annotated_bed     BED file of annotated isoforms, required by --annotation_reliant.
                    If this file is not provided, flair collapse will generate the
                    bedfile from the gtf. Eventually this argument will be removed. #提供isoforms注释的BED文件。
--range             Interval for which to collapse isoforms, formatted
                    chromosome:coord1-coord2 or tab-delimited; if a range is specified,
                    then the --reads argument must be a BAM file and --query must be
                    a sorted, bgzip-ed bed file. #isoforms合并间隔。

$ flair collapse -g genome.fa --gtf gene_annotations.gtf -q reads.flair_all_corrected.bed -r reads.fastq
--stringent --check_splice --generate_map --annotation_reliant generate

$ flair collapse -g genome.fa --gtf gene_annotations.gtf -q reads.flair_all_corrected.bed -r reads.fastq
--stringent --no_gtf_end_adjustment --check_splice --generate_map --trust_ends

usage: flair quantify -r reads_manifest.tsv -i isoforms.fa [options]

--isoforms          Fasta of Flair collapsed isoforms #最终合并的isoform序列文件，来自flair collapse。
--reads_manifest    Tab delimited file containing sample id, condition, batch,
                    reads.fq, where reads.fq is the path to the sample fastq file. #制表符分隔的样本id，实验条件分组，实验批次，测序数据（reads.fq）的路径。

sample1      condition1      batch1  mydata/sample1.fq
sample2      condition1      batch1  mydata/sample2.fq
sample3      condition1      batch1  mydata/sample3.fq
sample4      condition2      batch1  mydata/sample4.fq
sample5      condition2      batch1  mydata/sample5.fq
sample6      condition2      batch1  mydata/sample6.fq

-help               Show all options #帮助命令
--output            Name base for output files (default: flair.quantify). You
                    can supply an output directory (e.g. output/flair_quantify).  #指定输出文件前缀和路径。
--threads           Number of processors to use (default 4). #线程，默认为4。
--temp_dir          Directory to put temporary files. use ./ to indicate current
                    directory (default: python tempfile directory). #临时文件存放路径。
--sample_id_only    Only use sample id in output header instead of a concatenation
                    of id, condition, and batch. #在表达矩阵表头只显示样本名称，而不是id，分组，批次都显示。
--quality           Minimum MAPQ of read assignment to an isoform (default 1). #测序序列指定（归类）到isoform时的最小MAPQ，默认值为1。
--trust_ends        Specify if reads are generated from a long read method with
                    minimal fragmentation. #如果序列来自长序列建库方法（最小程度的打断）。
--generate_map      Create read-to-isoform assignment files for each sample. #生成序列对应isoform的文本文件，默认不指定。
--isoform_bed       isoform .bed file, must be specified if --stringent or
                    --check-splice is specified. #isoform的bed文件。如果指定--stringent和--check-splice，这必须提供。
--stringent         Supporting reads must cover 80% of their isoform and extend
                    at least 25 nt into the first and last exons. If those exons
                    are themselves shorter than 25 nt, the requirement becomes
                    'must start within 4 nt from the start' or 'end within 4 nt
                    from the end'. #支持序列必须都是全长（80%的覆盖率，第一个和最后一个外显子至少有25个碱基）。
--check_splice      Enforces coverage of 4 out of 6 bp around each splice site
                    and no insertions greater than 3 bp at the splice site. #至少覆盖剪切位点6个中的4个，插入序列不能大约3bp。

usage: flair_diffExp -q counts_matrix.tsv --out_dir out_dir [options]

--counts_matrix     Tab-delimited isoform count matrix from flair quantify #flair定量表达矩阵。
--out_dir           Output directory for tables and plots. #输出文件夹路径。

--help              Show this help message and exit #帮助。
--threads           Number of threads for parallel DRIMSeq. #运行DRIMseq的线程数。
--exp_thresh        Read count expression threshold. Isoforms in which both
                    conditions contain fewer than E reads are filtered out (Default E=10) #isoform表达count数阈值，低于此值则舍去。默认值为10。
--out_dir_force     Specify this argument to force overwriting of files in
                    an existing output directory #输出路径。

usage: flair_diffSplice -i isoforms.bed -q counts_matrix.tsv [options]

--isoforms          Isoforms in bed format from Flair collapse. #isoform的bed文件。
--counts_matrix     Tab-delimited isoform count matrix from Flair quantify. #isoform表达矩阵
--out_dir           Output directory for tables and plots. #输出文件夹路径。

--help              Show all options. #帮助选项
--threads           Number of processors to use (default 4). #使用线程，默认为4》
--test              Run DRIMSeq statistical testing. #使用DRIMSeq进行统计学分析。
--drim1             The minimum number of samples that have coverage over an
                    AS event inclusion/exclusion for DRIMSeq testing; events
                    with too few samples are filtered out and not tested (6). #对可变剪切事件（保留和排除）有覆盖度的最小样本数。
--drim2             The minimum number of samples expressing the inclusion of
                    an AS event; events with too few samples are filtered out
                    and not tested (3). #包含保留可变剪切事件的最小样本数。
--drim3             The minimum number of reads covering an AS event
                    inclusion/exclusion for DRIMSeq testing, events with too
                    few samples are filtered out and not tested (15). #对可变剪切事件（保留和排除）有覆盖度的最小read数。
--drim4             The minimum number of reads covering an AS event inclusion
                    for DRIMSeq testing, events with too few samples are
                    filtered out and not tested (5).#包含保留可变剪切事件的最小read数。
--batch             If specified with --test, DRIMSeq will perform batch correction. #DRIMSeq可以进行批次校正。
--conditionA        Specify one condition corresponding to samples in the
                    counts_matrix to be compared against condition2; by default,
                    the first two unique conditions are used. This implies --test. #指定差异分析比对组。
--conditionB        Specify another condition corresponding to samples in the
                    counts_matrix to be compared against conditionA. #指定差异分析比对组。
--out_dir_force     Specify this argument to force overwriting of files in an
                    existing output directory #输出路径。

a3ss_feature_id     coordinate                  sample1 sample2 ... isoform_ids
inclusion_chr1:80   chr1:80-400_chr1:80-450     75.0    35.0    ... a,e
exclusion_chr1:80   chr1:80-400_chr1:80-450     3.0     13.0    ... c
inclusion_chr1:500  chr1:500-650_chr1:500-700   4.0     18.0    ... d
exclusion_chr1:500  chr1:500-650_chr1:500-700   70.0    17.0    ... e

a3ss_feature_id     coordinate                  sample1 sample2 ... isoform_ids
inclusion_chr1:80   chr1:80-400_chr1:80-450     75.0    35.0    ... a,e
exclusion_chr1:80   chr1:80-400_chr1:80-450     3.0     13.0    ... c
inclusion_chr1:500  chr1:500-650_chr1:500-700   4.0     18.0    ... d
exclusion_chr1:500  chr1:500-650_chr1:500-700   70.0    17.0    ... e

a3ss_feature_id     coordinate                  sample1 sample2 ... isoform_ids
inclusion_chr1:80   chr1:80-400_chr1:80-450     75.0    35.0    ... a,e
exclusion_chr1:80   chr1:80-400_chr1:80-450     3.0     13.0    ... c
inclusion_chr1:500  chr1:500-650_chr1:500-700   4.0     18.0    ... d
exclusion_chr1:500  chr1:500-650_chr1:500-700   70.0    17.0    ... e