Figure FAIRE Procedure (A) The FAIRE procedure described in the text is shown on the left, while preparation of the reference or input sample is shown on the right. The DNA recovered from he aqueous phase of each extraction can then be used to identify sites of open chromatin using qPCR, tiling microarrays, or high-throughput sequencing applications. (B) For qPCR, a series of primers, depicted as convergent arrows, are designed to span a genomic region of interest. Sites of open chromatin are highlighted in blue, with qPCR results depicted above. Amplicons that span or are near the boundaries of open chromatin often result in lower relative enrichment due to shearing of DNA fragments, as shown by asterisks. (C) Microarrays. Typically we use highresolution microarrays that tile either regions of interest or the entire genome of an organism with 50 to 70 bp oligonucleotides. (D) High-throughput sequencing technologies can be used to map the DNA fragments back to the reference genome.【Giresi P G, Lieb J D. Isolation of active regulatory elements from eukaryotic chromatin using FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements)[J]. Methods, 2009, 48(3):233-239.】

Comparison of experimental protocols.Experiments to detect different aspects of DNA-binding proteins share many of the same steps; simplified schematics of the main steps are shown. a | Chromatin immunoprecipitation followed by sequencing (ChIP–seq) for DNA-binding proteins such as transcription factors. Recent variations on the standard protocol include using endonuclease digestion instead of sonication (ChIP–exo) to increase the resolution of binding-site detection and to eliminate contaminating DNA, and DNA amplification after ChIP for samples with limited cells. b | ChIP–seq for histone modifications uses micrococcal nuclease (MNase) digestion to fragment DNA and can also now be run on low-quantity samples when combined with the additional post-ChIP amplification. c | DNase–seq relies on digestion by the DNaseI nuclease to identify regions of nucleosome-depleted open chromatin where there are binding sites for all types of factors, but it cannot identify what specific factors are bound. d | Formaldehyde-assisted identification of regulatory elements (FAIRE–seq) similarly identifies nucleosome-depleted regions by extracting fragmented DNA that is not crosslinked to nucleosomes. LinDA, single-tube linear DNA amplification; T7, T7 phage RNA polymerase.【Furey, Terrence S . ChIP–seq and beyond: new and improved methodologies to detect and characterize protein–DNA interactions[J]. Nature Reviews Genetics, 2012, 13(12):840-852.】

(1)按生产商说明,制备测序文库。这里我们推荐100~200 ng的 FAIRE DNA样品.首先使用 Agencourt AMPure XP beads 试剂盒进行两轮纯化与回收,然后进行18个循环的PCR扩增,筛选200-~500bp的DNA建立最终文库。另外,也可以用TruSeq kit(Illumina)试剂盒来制备测序文库。关键步骤:保证至少3×10’读段(reads),才可以确保测序深度和广度
(2)运用诸如TagDust(的算法,通过适配器过滤去除测序序列,清洗文库.我们设定错误发现率为0.001,关键步骤:在清洗文库步骤中,估计会过滤掉大概0.1~0.2%的读段.如果过滤部分高达10%,则说明文库的质量不达标.
(3)使用FASTX-Toolkit 算法(http://hannonlab.cshl.edu/fastx_toolkit/) 来评估文库质量,比如说置信值(confidence score)),核苷酸分布(nucleotide distribution).关键步骤:相对均一的碱基分布非常重要,在每个读段中都保证有四种核苷酸。核苷酸的误用,特殊序列的重复出现和序列质量的不稳定性,都有可能导致测序文库更为复杂以及测序错误增加。
(4)使用诸如Bowtie6这样的算法,将高质量的读段映射(mapping)到参考基因组上。大都采取默认参数,可允许的比对数量的最大值限定为4.当有多种比对(alignment)结果存在时,Bowtie算法必定会选择得分最高的比对结果。对于大部分的基因组和实验来说,大约有75~85%的测序读設可以被成功比对.
(5)数据可视化以及寻找发观相对于背景有明显富集的检测区域(使用ZINB算法(60).
(6)使用IDR算法(6기来评估交联-折叠相关系数I45].