4.对原始数据进行fastqc质控

#设置参考基因相关文件变量，方便后续使用

REF=$refdir/Arabidopsis_thaliana.TAIR10.dna.chromosome.4.fa

BWA_INDEX=$refdir/Arabidopsis_thaliana.TAIR10.dna.chromosome.4.fa

GFF=$refdir/Arabidopsis_thaliana.TAIR10.47.chromosome.4.gff3

GTF=$refdir/Arabidopsis_thaliana.TAIR10.47.chromosome.4.gtf

INDEX_FAI=$refdir/Arabidopsis_thaliana.TAIR10.dna.chromosome.4.fa.fai

PICARD_DICT=$refdir/Arabidopsis_thaliana.TAIR10.dna.chromosome.4.dict



(base) [niujiajia@localhost ref]$ cd $workdir

mkdir 1.fastqc

fastqc $datadir/*.gz -o $workdir/1.fastqc

(base) [niujiajia@localhost work]$ cd 1.fastqc/

(base) [niujiajia@localhost 1.fastqc]$ ll

总用量 12072

-rw-rw-r--. 1 niujiajia niujiajia 609995 10月 24 22:06 p1_r1_fastqc.html

-rw-rw-r--. 1 niujiajia niujiajia 909592 10月 24 22:06 p1_r1_fastqc.zip

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#1.安装fastp

conda install -c "bioconda/label/cf201901" fastp



#2.数据质控：对原始序列进行去接头，删除低质量的reads等等

cd $workdir #回到工作目录

mkdir 2.data_qc

cd 2.data_qc



for i in p1 p2 pool1 pool2; do

echo "fastp -t 4 -q 10 -u 50 -l 10 -i $datadir/${i}_r1.fq.gz -I $datadir/${i}_r2.fq.gz -o ${i}_1.clean.fq.gz -O ${i}_2.clean.fq.gz -h ${i}.html -j ${i}.json"

fastp fastp -t 4 -q 10 -u 50 -l 10 --length_limit 0 -i $datadir/${i}_r1.fq.gz -I $datadir/${i}_r2.fq.gz -o ${i}_1.clean.fq.gz -O ${i}_2.clean.fq.gz -h ${i}.html -j ${i}.json

done



python $scriptdir/qc_stat.py -d $workdir/2.data_qc/ -o $workdir/2.data_qc/ -p all_sample_qc



(base) [niujiajia@localhost 2.data_qc]$ ll

总用量 557080

-rw-rw-r--. 1 niujiajia niujiajia 340 10月 24 23:34 all_sample_qc.tsv

-rw-rw-r--. 1 niujiajia niujiajia 71356114 10月 24 23:31 p1_1.clean.fq.gz

-rw-rw-r--. 1 niujiajia niujiajia 73151726 10月 24 23:31 p1_2.clean.fq.gz

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-rw-rw-r--. 1 niujiajia niujiajia 128043 10月 24 23:31 p1.json

-rw-rw-r--. 1 niujiajia niujiajia 71446954 10月 24 23:31 p2_1.clean.fq.gz

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-rw-rw-r--. 1 niujiajia niujiajia 67750448 10月 24 23:31 pool2_1.clean.fq.gz

-rw-rw-r--. 1 niujiajia niujiajia 72439232 10月 24 23:31 pool2_2.clean.fq.gz

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-rw-rw-r--. 1 niujiajia niujiajia 128548 10月 24 23:31 pool2.json

fastp功能

usage: fastp [options] ... 
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 [=])
  -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                         stream passing-filters reads to STDOUT. This option will result in interleaved FASTQ output for paired-end input. Disabled by defaut.
      --interleaved_in                 indicate that <in1> is an interleaved FASTQ which contains both read1 and read2. Disabled by defaut.
      --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.
  -V, --verbose                        output verbose log information (i.e. when every 1M reads are processed).
  -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 [=])
  -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])
  -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])
  -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
  -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])
  -5, --cut_by_quality5                enable per read cutting by quality in front (5'), default is disabled (WARNING: this will interfere deduplication for both PE/SE data)
  -3, --cut_by_quality3                enable per read cutting by quality in tail (3'), default is disabled (WARNING: this will interfere deduplication for SE data)
  -W, --cut_window_size                the size of the sliding window for sliding window trimming, default is 4 (int [=4])
  -M, --cut_mean_quality               the bases in the sliding window with mean quality below cutting_quality will be cut, default is Q20 (int [=20])
  -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])
  -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])
  -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_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])
  -c, --correction                     enable base correction in overlapped regions (only for PE data), default is disabled
      --overlap_len_require            the minimum length of the overlapped region for overlap analysis based adapter trimming and correction. 30 by default. (int [=30])
      --overlap_diff_limit             the maximum difference of the overlapped region for overlap analysis based adapter trimming and correction. 5 by default. (int [=5])
  -U, --umi                            enable unique molecular identifer (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])
  -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])
  -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])
  -w, --thread                         worker thread number, default is 2 (int [=2])
  -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                           print this message