Fastp:

```
fastp --thread 4 -l 50 -q 20 -u 20 --adapter_sequence ${adapter_sequence} --adapter_sequence_r2 ${adapter_sequence_r2} -- -i  ${read1} -I ${read2} -o ${sample_id}_R1.fastq.gz -O ${sample_id}_R2.fastq.gz -j ${sample_id}.json -h ${sample_id}.html
```



```
usage: fastp -i <in1> -o <out1> [-I <in1> -O <out2>] [options...]
options:
  # I/O options
  -i, --in1                          read1 input file name (string)
  -o, --out1                         read1 output file name (string [=])
  -I, --in2                          read2 input file name (string [=])
  -O, --out2                           read2 output file name (string [=])
      --unpaired1                      for PE input, if read1 passed QC but read2 not, it will be written to unpaired1. Default is to discard it. (string [=])
      --unpaired2                      for PE input, if read2 passed QC but read1 not, it will be written to unpaired2. If --unpaired2 is same as --unpaired1 (default mode), both unpaired reads will be written to this same file. (string [=])
      --failed_out                     specify the file to store reads that cannot pass the filters. (string [=])
      --overlapped_out                 for each read pair, output the overlapped region if it has no any mismatched base. (string [=])
  -m, --merge                          for paired-end input, merge each pair of reads into a single read if they are overlapped. The merged reads will be written to the file given by --merged_out, the unmerged reads will be written to the files specified by --out1 and --out2. The merging mode is disabled by default.
      --merged_out                     in the merging mode, specify the file name to store merged output, or specify --stdout to stream the merged output (string [=])
      --include_unmerged               in the merging mode, write the unmerged or unpaired reads to the file specified by --merge. Disabled by default.
  -6, --phred64                      indicate the input is using phred64 scoring (it'll be converted to phred33, so the output will still be phred33)
  -z, --compression                  compression level for gzip output (1 ~ 9). 1 is fastest, 9 is smallest, default is 4. (int [=4])
      --stdin                          input from STDIN. If the STDIN is interleaved paired-end FASTQ, please also add --interleaved_in.
      --stdout                         output passing-filters reads to STDOUT. This option will result in interleaved FASTQ output for paired-end input. Disabled by default.
      --interleaved_in                 indicate that <in1> is an interleaved FASTQ which contains both read1 and read2. Disabled by default.
      --reads_to_process             specify how many reads/pairs to be processed. Default 0 means process all reads. (int [=0])
      --dont_overwrite               don't overwrite existing files. Overwritting is allowed by default.
      --fix_mgi_id                     the MGI FASTQ ID format is not compatible with many BAM operation tools, enable this option to fix it.
  
  # adapter trimming options
  -A, --disable_adapter_trimming     adapter trimming is enabled by default. If this option is specified, adapter trimming is disabled
  -a, --adapter_sequence               the adapter for read1. For SE data, if not specified, the adapter will be auto-detected. For PE data, this is used if R1/R2 are found not overlapped. (string [=auto])
      --adapter_sequence_r2            the adapter for read2 (PE data only). This is used if R1/R2 are found not overlapped. If not specified, it will be the same as <adapter_sequence> (string [=])
      --adapter_fasta                  specify a FASTA file to trim both read1 and read2 (if PE) by all the sequences in this FASTA file (string [=])
      --detect_adapter_for_pe          by default, the adapter sequence auto-detection is enabled for SE data only, turn on this option to enable it for PE data.
    
  # global trimming options
  -f, --trim_front1                    trimming how many bases in front for read1, default is 0 (int [=0])
  -t, --trim_tail1                     trimming how many bases in tail for read1, default is 0 (int [=0])
  -b, --max_len1                       if read1 is longer than max_len1, then trim read1 at its tail to make it as long as max_len1. Default 0 means no limitation (int [=0])
  -F, --trim_front2                    trimming how many bases in front for read2. If it's not specified, it will follow read1's settings (int [=0])
  -T, --trim_tail2                     trimming how many bases in tail for read2. If it's not specified, it will follow read1's settings (int [=0])
  -B, --max_len2                       if read2 is longer than max_len2, then trim read2 at its tail to make it as long as max_len2. Default 0 means no limitation. If it's not specified, it will follow read1's settings (int [=0])

  # polyG tail trimming, useful for NextSeq/NovaSeq data
  -g, --trim_poly_g                  force polyG tail trimming, by default trimming is automatically enabled for Illumina NextSeq/NovaSeq data
      --poly_g_min_len                 the minimum length to detect polyG in the read tail. 10 by default. (int [=10])
  -G, --disable_trim_poly_g          disable polyG tail trimming, by default trimming is automatically enabled for Illumina NextSeq/NovaSeq data

  # polyX tail trimming
  -x, --trim_poly_x                    enable polyX trimming in 3' ends.
      --poly_x_min_len                 the minimum length to detect polyX in the read tail. 10 by default. (int [=10])
  
  # per read cutting by quality options
  -5, --cut_front                      move a sliding window from front (5') to tail, drop the bases in the window if its mean quality < threshold, stop otherwise.
  -3, --cut_tail                       move a sliding window from tail (3') to front, drop the bases in the window if its mean quality < threshold, stop otherwise.
  -r, --cut_right                      move a sliding window from front to tail, if meet one window with mean quality < threshold, drop the bases in the window and the right part, and then stop.
  -W, --cut_window_size                the window size option shared by cut_front, cut_tail or cut_sliding. Range: 1~1000, default: 4 (int [=4])
  -M, --cut_mean_quality               the mean quality requirement option shared by cut_front, cut_tail or cut_sliding. Range: 1~36 default: 20 (Q20) (int [=20])
      --cut_front_window_size          the window size option of cut_front, default to cut_window_size if not specified (int [=4])
      --cut_front_mean_quality         the mean quality requirement option for cut_front, default to cut_mean_quality if not specified (int [=20])
      --cut_tail_window_size           the window size option of cut_tail, default to cut_window_size if not specified (int [=4])
      --cut_tail_mean_quality          the mean quality requirement option for cut_tail, default to cut_mean_quality if not specified (int [=20])
      --cut_right_window_size          the window size option of cut_right, default to cut_window_size if not specified (int [=4])
      --cut_right_mean_quality         the mean quality requirement option for cut_right, default to cut_mean_quality if not specified (int [=20])
  
  # quality filtering options
  -Q, --disable_quality_filtering    quality filtering is enabled by default. If this option is specified, quality filtering is disabled
  -q, --qualified_quality_phred      the quality value that a base is qualified. Default 15 means phred quality >=Q15 is qualified. (int [=15])
  -u, --unqualified_percent_limit    how many percents of bases are allowed to be unqualified (0~100). Default 40 means 40% (int [=40])
  -n, --n_base_limit                 if one read's number of N base is >n_base_limit, then this read/pair is discarded. Default is 5 (int [=5])
  -e, --average_qual                 if one read's average quality score <avg_qual, then this read/pair is discarded. Default 0 means no requirement (int [=0])

  
  # length filtering options
  -L, --disable_length_filtering     length filtering is enabled by default. If this option is specified, length filtering is disabled
  -l, --length_required              reads shorter than length_required will be discarded, default is 15. (int [=15])
      --length_limit                 reads longer than length_limit will be discarded, default 0 means no limitation. (int [=0])

  # low complexity filtering
  -y, --low_complexity_filter          enable low complexity filter. The complexity is defined as the percentage of base that is different from its next base (base[i] != base[i+1]).
  -Y, --complexity_threshold           the threshold for low complexity filter (0~100). Default is 30, which means 30% complexity is required. (int [=30])

  # filter reads with unwanted indexes (to remove possible contamination)
      --filter_by_index1               specify a file contains a list of barcodes of index1 to be filtered out, one barcode per line (string [=])
      --filter_by_index2               specify a file contains a list of barcodes of index2 to be filtered out, one barcode per line (string [=])
      --filter_by_index_threshold      the allowed difference of index barcode for index filtering, default 0 means completely identical. (int [=0])

  # base correction by overlap analysis options
  -c, --correction                   enable base correction in overlapped regions (only for PE data), default is disabled
      --overlap_len_require            the minimum length to detect overlapped region of PE reads. This will affect overlap analysis based PE merge, adapter trimming and correction. 30 by default. (int [=30])
      --overlap_diff_limit             the maximum number of mismatched bases to detect overlapped region of PE reads. This will affect overlap analysis based PE merge, adapter trimming and correction. 5 by default. (int [=5])
      --overlap_diff_percent_limit     the maximum percentage of mismatched bases to detect overlapped region of PE reads. This will affect overlap analysis based PE merge, adapter trimming and correction. Default 20 means 20%. (int [=20])

  # UMI processing
  -U, --umi                          enable unique molecular identifier (UMI) preprocessing
      --umi_loc                      specify the location of UMI, can be (index1/index2/read1/read2/per_index/per_read, default is none (string [=])
      --umi_len                      if the UMI is in read1/read2, its length should be provided (int [=0])
      --umi_prefix                   if specified, an underline will be used to connect prefix and UMI (i.e. prefix=UMI, UMI=AATTCG, final=UMI_AATTCG). No prefix by default (string [=])
      --umi_skip                       if the UMI is in read1/read2, fastp can skip several bases following UMI, default is 0 (int [=0])

  # overrepresented sequence analysis
  -p, --overrepresentation_analysis    enable overrepresented sequence analysis.
  -P, --overrepresentation_sampling    One in (--overrepresentation_sampling) reads will be computed for overrepresentation analysis (1~10000), smaller is slower, default is 20. (int [=20])

  # reporting options
  -j, --json                         the json format report file name (string [=fastp.json])
  -h, --html                         the html format report file name (string [=fastp.html])
  -R, --report_title                 should be quoted with ' or ", default is "fastp report" (string [=fastp report])
  
  # threading options
  -w, --thread                       worker thread number, default is 2 (int [=2])
  
  # output splitting options
  -s, --split                        split output by limiting total split file number with this option (2~999), a sequential number prefix will be added to output name ( 0001.out.fq, 0002.out.fq...), disabled by default (int [=0])
  -S, --split_by_lines               split output by limiting lines of each file with this option(>=1000), a sequential number prefix will be added to output name ( 0001.out.fq, 0002.out.fq...), disabled by default (long [=0])
  -d, --split_prefix_digits          the digits for the sequential number padding (1~10), default is 4, so the filename will be padded as 0001.xxx, 0 to disable padding (int [=4])
  
  # help
  -?, --help                         print this message
```



hisat2

```
 hisat2 -t -p $nt -x ${idx}/${idx_prefix} -1 ${Trim_R1} -2 ${Trim_R2} -S ${sample_id}.sam  --un-conc-gz ${sample_id}_un.fq.gz
```



```
Usage

hisat [options]* -x <hisat-idx> {-1 <m1> -2 <m2> | -U <r> | --sra-acc <SRA accession number>} [-S <hit>]
Main arguments

-x <hisat-idx>	The basename of the index for the reference genome. 

-1 <m1>；-2 <m2>


Spliced alignment options

--pen-cansplice <int>
为每对规范的剪接位点（例如GT / AG）设置惩罚。默认值：0

--pen-noncansplice <int>
设置每对非规范剪接位点（例如非GT / AG）的惩罚。默认值：3。

--pen-intronlen <func>
设置长内含子的罚分，因此与较短的内含子相比，较短的内含子优先。默认值：G，-8,1

--min-intronlen <int>
设置最小内含子长度。默认值：20

--max-intronlen <int>
设置最大内含子长度。默认值：500000

Paired-end options

-I/--minins <int>
有效的配对末端比对的最小片段长度。例如，如果-I 60已指定并且一对末端比对包含两个处于适当方向的20 bp比对，且两者之间有20 bp的间隔，则认为该比对是有效的（只要-X也可以满足）。在这种情况下，19bp的缺口将无效。如果使用修剪选项-3或-5也使用修剪选项，则将-I约束应用于未修剪的配合。

-I和之间的差异越大-X，HISAT运行的速度就越慢。这是因为在更大的差异之间，-I并且-X要求HISAT扫描更大的窗口以确定是否存在一致的对齐方式。对于典型的片段长度范围（200至400个核苷酸），HISAT非常有效。

Default: 0 (essentially imposing no minimum)

-X/--maxins <int>
有效的配对末端比对的最大片段长度。例如，如果-X 100已指定并且一对末端比对包含两个正确方向的20 bp比对，且两者之间有60 bp的间隔，则认为该比对是有效的（只要-I也可以满足）。在这种情况下，61bp的缺口将无效。如果使用-3或修剪选项-5，则-X约束将应用于未修剪的配合，而不是修剪的配合。

-I和之间的差异越大-X，HISAT运行的速度就越慢。这是因为在更大的差异之间，-I并且-X要求HISAT扫描更大的窗口以确定是否存在一致的对齐方式。对于典型的片段长度范围（200至400个核苷酸），HISAT非常有效。

默认值：500。

Default: 500.

```

samtools

```
samtools view -bS ${sam} > ${bam}
samtools sort -m 1000000000 ${bam} -o ${sorted_bam}
samtools index ${sorted_bam}
samtools stats -i 8000 ${sorted_bam} |grep ^IS|cut -f 2- > ${sample_id}.ins_size
```

```
-i，--insert-size INT
最大插入尺寸[8000]
samtools view -bs 42.1 ${bam} > ${10percent_bam}
samtools view -bs {ratio} ${bam} > ${percent_bam}
```

stringtie

```
stringtie -e -B -p $nt -G ${gtf} -o ballgown/${sample_id}/${sample_id}.gtf -C ${sample_id}.cov.ref.gtf -A ${sample_id}.gene.abundance.txt ${bam} -g ${sample_id}_genecount.csv
```

```
-f 将预测的转录本的最小同工型丰度设置为在给定基因座处组装的最丰富的转录本的一部分。低丰度转录物通常是加工的转录物的不完全剪接的前体的产物。默认值：0.01
-m <整数>	设置预测成绩单所允许的最小长度。默认值：200
-a <int>	没有拼接的接头在两端至少与该数量的碱基对齐，这些接头被过滤掉。默认值：10
-M <0.0-1.0>	设置允许在给定基因座处存在的多核苷酸位置映射的读数的最大分数。默认值：0.95
stringtie -e -B -p $nt -f ${minimum_isoform_abundance} -m ${minimum_length_allowed_for_the_predicted_transcripts} -a ${Junctions_no_spliced_reads} -M ${maximum_fraction_of_muliplelocationmapped_reads} -G ${gtf} -o ballgown/${sample_id}/${sample_id}.gtf -C ${sample_id}.cov.ref.gtf -A ${sample_id}.gene.abundance.txt ${bam} -g ${sample_id}_genecount.csv
```