在制备测序文库时，经常会增加PCR的步骤来扩增DNA片段。如何评估PCR的效果和影响，本文主要分享ENCODE中针对ChIP-Seq和ATAC-Seq标准来说明。

    ENCODE中主要通过三个参数来反应Library Complexity：PCB1，PBC2和NRF。以下分别介绍各自的定义：
    PCR Bottlenecking Coefficient 1 (PBC1)

    • PBC1=M1/M_DISTINCT where
      • M1: number of genomic locations where exactly one read maps uniquely
      • M_DISTINCT: number of distinct genomic locations to which some read maps uniquely

    PCR Bottlenecking Coefficient 2 (PBC2)

    • PBC2= M1/M2 where
      • M1: number of genomic locations where only one read maps uniquely
      • M2: number of genomic locations where two reads map uniquely

    Non-Redundant Fraction (NRF) - Number of distinct uniquely mapping reads (i.e. after removing duplicates) / Total number of reads.

    如何计算这三个数值呢，在我们拿到比对的结果后（SAM或者BAM文件）
    对于single end测序，以align.bam为例：

    1. bedtools bamtobed -i align.bam | \
    2.     awk \'BEGIN{OFS="\t"}{print $1,$2,$3,$6}\' | \
    3.     grep -v \'chrM\' | sort | uniq -c | \
    4.     awk \'BEGIN{mt=0;m0=0;m1=0;m2=0} ($1==1){m1=m1 1} ($1==2){m2=m2 1} {m0=m0 1} {mt=mt $1} END{m1_m2=-1.0; if(m2>0) m1_m2=m1/m2; printf "%d\t%d\t%d\t%d\t%f\t%f\t%f\n",mt,m0,m1,m2,m0/mt,m1/m0,m1_m2}\' > pbc_qc.txt

    对于pair-end测序，以align.bam为例：

    1. bedtools bamtobed -bedpe -i align.bam | \
    2.     awk \'BEGIN{OFS="\t"}{print $1,$2,$4,$6,$9,$10}\' | \
    3.     grep -v \'chrM\' | sort | uniq -c | \
    4.     awk \'BEGIN{mt=0;m0=0;m1=0;m2=0} ($1==1){m1=m1 1} ($1==2){m2=m2 1} {m0=m0 1} {mt=mt $1} END{m1_m2=-1.0; if(m2>0) m1_m2=m1/m2; printf "%d\t%d\t%d\t%d\t%f\t%f\t%f\n",mt,m0,m1,m2,m0/mt,m1/m0,m1_m2}\' > pbc_qc.txt

    最后文件中包含7列，分别为：
    1）TotalReadPairs
    2）DistinctReadPairs
    3）OneReadPair
    4）TwoReadPairs
    5）NRF=Distinct/Total
    6）PBC1=OnePair/Distinct
    7）PBC2=OnePair/TwoPair

    对于ChIP-seq结果解读:
    image.png
    对于ATAC-Seq结果解读:
    image.png