基本概述：

bedtools subtract 通俗的说，得到 A - B 的区段。如果在A中发现了B区段，就把 B 扣除，通过不同的参数，扣除的标准不一样。其中，参数 -A 可以达成 Remove features with any overlap 的效果（第四行）。

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使用方法：

bedtools subtract [OPTIONS] -a <BED/GFF/VCF> -b <BED/GFF/VCF>

或者简写成

subtractBed [OPTIONS] -a <BED/GFF/VCF> -b <BED/GFF/VCF>

参数一览

Option	Description
-f	Minimum overlap required as a fraction of A. Default is 1E-9 (i.e. 1bp).
-F	Minimum overlap required as a fraction of B. Default is 1E-9 (i.e., 1bp).
-r	Require that the fraction of overlap be reciprocal for A and B. In other words, if -f is 0.90 and -r is used, this requires that B overlap at least 90% of A and that A also overlaps at least 90% of B.
-e	Require that the minimum fraction be satisfied for A _OR_ B. In other words, if -e is used with -f 0.90 and -F 0.10 this requires that either 90% of A is covered OR 10% of B is covered. Without -e, both fractions would have to be satisfied.
-s	Force “strandedness”. That is, only report hits in B that overlap A on the same strand. By default, overlaps are reported without respect to strand.
-S	Require different strandedness. That is, only report hits in B that overlap A on the _opposite_ strand. By default, overlaps are reported without respect to strand.
-A	Remove entire feature if any overlap. That is, by default, only subtract the portion of A that overlaps B. Here, if any overlap is found (or -f amount), the entire feature is removed.
-N	Same as -A except when used with -f, the amount is the sum of all features (not any single feature).

默认参数：

$ cat A.bed
chr1  10   20
chr1  100  200$ cat B.bed
chr1  0    30
chr1  180  300$ bedtools subtract -a A.bed -b B.bed
chr1  100  180

A     10 - 20       100 - 200
B 0-30                     180-300
默认状态 A 第一个区段全扣除，第二个区段180之后的序列扣除，最终剩下100-180.

-f 重叠参数

$ cat A.bed
chr1  100  200$ cat B.bed
chr1  180  300$ bedtools subtract -a A.bed -b B.bed -f 0.10
chr1  100  180$ bedtools subtract -a A.bed -b B.bed -f 0.80
chr1  100  200

f 这个参数，如果B的区段覆盖度不满足这个参数，就不被扣除。0.10 扣了，0.80 没扣。

-s 考虑特征（正反链）

$ cat A.bed
chr1  100  200    a1  1   +$ cat B.bed
chr1  80   120    b1  1   +
chr1  180  300    b2  1   -$ bedtools subtract -a A.bed -b B.bed -S
chr1  100  180    a1  1   +

-A Remove features with any overlap：

重点来了，-A 模式下，那么 B 里面包含 A 里 包含一个 1个BP，也是整段删除。“”沾边删” 模式

$ cat A.bed
chr1  100  200$ cat B.bed
chr1  180  300$ bedtools subtract -a A.bed -b B.bed
chr1  100  180$ bedtools subtract -a A.bed -b B.bed -A