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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