主流工具:

拿到测序数据的第一步就是做质量控制

fqcheck之后得到的结果:

FASTQ 数据质量统计工具-LMLPHP

它会统计每条reads,按read 1-100位点计算每个位置的ACGTN含量,以及0-41质量值的个数

最终会得到整体的错误率,GC,Q20,Q30

the default quality shift value is: -64, 27877224 sequences, 2787722400 total length, Max length:100, average length:100.00
Standard deviations at 0.25: total 0.00%, per base 0.01%
···
Error Rate %GC Q20 Q30
0.61 48.35 96.26 89.88

 

adapter.list

#reads_id   reads_len   reads_start   reads_end   adapter_id   adapter_len   adapter_start   adapter_end   align_len   mismatch
FCD0JN9ACXX:6:1101:13637:2052#AGAGATCT/1 100 57 90 iPE-3+ 34 0 33 34 13
FCD0JN9ACXX:6:1101:15321:2200#AGAGATCT/1 100 53 86 iPE-3+ 34 0 33 34 0
FCD0JN9ACXX:6:1101:5318:2346#AGAGATCT/1 100 60 93 iPE-3+ 34 0 33 34 0
FCD0JN9ACXX:6:1101:5745:2411#AGAGATCT/1 100 64 97 iPE-3+ 34 0 33 34 0
FCD0JN9ACXX:6:1101:13286:2320#AGAGATCT/1 100 89 99 iPE-3+ 34 0 10 11 2
FCD0JN9ACXX:6:1101:15982:2390#AGAGATCT/1 100 80 99 iPE-3+ 34 0 19 20 0

接头序列,一般都要去掉

Illumina Adapter Sequences Document (1000000002694 v01)

 

过滤

使用SOAPnuke

SOAPnuke filter -l 20 -q 0.5 -n 0.1 -d -i -Q 1 -5 0 -1 1.fq.gz -2 2.fq.gz -f 1.adapter.list.gz -r 2.adapter.list.gz $tile -o 16_1.fq -D 16_2.fq -c 21

过滤后的reads同样要做质量统计

fqcheck -r 16_1.fq.gz -c 16_1.fqcheck

之后还会写个脚本作 fqcheck_distribute 分析

 

过滤后统计 FilterStat

得到

Type    Raw data        Clean data
Number of Reads 52293338 48926594
Data Size 5229333800 4892659400
N of fq1 146135 35060
N of fq2 399754 16287
GC(%) of fq1 45.53 45.36
GC(%) of fq2 45.58 45.39
Q20(%) of fq1 97.03 97.99
Q20(%) of fq2 92.83 95.92
Q30(%) of fq1 91.66 93.58
Q30(%) of fq2 86.07 89.72
Discard Reads related to N 24406
Discard Reads related to low qual 2917634
Discard Reads related to Adapter 135524

catRS

drawPizza

 

参考:

质量值体系 Phred33 和 Phred 64 的由来 及其在质量控制中的实际影响 - Part 2

05-18 08:34