Development And Application Of Single-strand-based NGS Library Construction Methods

During the last decade,the most striking achievement about cancer is that cancer could be induced by epigenetic disorder as abnormal of genetic.Chromatin accessibility as an important feature of epigenetic play key roles in initiation and development process of cancer.Open chromatin provide binding sites for transcription factor,which regulated the gene expression during cancer process.For these reasons,it is meaningful for identification of chromatin accessibility of cancer cell not only in recognizing the mechanism of cancer but also in treating cancer.Cell free DNA(cfDNA)as an important sample of biopsy test,server essential function in IVD(in vitro diagnosis),especially in cancer related mutation test.Using cfDNA to identify the chromatin state in cancer patients,could greatly extend the application area of cfDNA from genetic to epigenetic.And NGS play key roles in all of these area.In this study,multiple research were performed which were related above aspects,and the main achievement were listed below.1.Establishment of novel high throughput chromatin open state identification method– SALPNext-generation sequencing(NGS)is fundamental to the current biological and biomedical research.Construction of sequencing library is a key step of NGS.Therefore,various library construction methods have been explored.However,the current methods are still limited by some shortcomings.This study developed a new NGS library construction method,Single strand Adaptor Library Preparation(SALP),by using a novel single strand adaptor(SSA).SSA is a double-stranded oligonucleotide with a 3?overhang of 3 random nucleotides,which can be efficiently ligated to the 3?end of single strand DNA by T4 DNA ligase.SALP can be started with any denatured DNA fragments such as those sheared by Tn5 tagmentation,enzyme digestion and sonication.When started with Tn5-tagmented chromatin,SALP can overcome a key limitation of ATAC-seq and become a high-throughput NGS library construction method,SALP-seq,which can be used to comparatively characterize the chromatin openness state of multiple cells unbiasly.In this way,this study successfully characterized the comparative chromatin openness states of four different cell lines,including GM12878,HepG2,HeLa and293T,with SALP-seq.Similarly,this study also successfully characterized the chromatin openness states of HepG2 cells with SALP-seq by using 10~5 to 500 cells.Besides,using SALP-seq the chromatin state in different cell lines of super enhancer of MYC and TERT were also analysis,which showed significant cell type specificity.Based on the SALP-seq results of 4cell lines,totally 137 potential cancer related gene were identified.As a result,SALP developed in this research with the feature of high efficiency and easy-used could be applied in multiple areas.2.Comparison of chromatin open state of different cell type using SALPAbnormal of epigenetic is the common feature of human cancer,chromatin open state is an important part of epigenetics.For this reason,the chromatin openness of multiple cell lines including PANC-1,A549,HT29,SKOV3,SiHa,C-33A,HL7702,MRC-5,Hepa1-6 and normal liver cell from BALB/C mouse were identified with SALP-seq.Combined with GM12878,HepG2 and HeLa SALP-seq data,comparison were performed in several aspect.Significant chromatin state difference were discovered between cancer and normal cell lines,different kinds of cancer cell lines,two different normal cell lines,three different cervix cancer subtypes,and normal/cancer cells origin from same tissues.The chromatin state and transcription factor difference between normal and cancer cell lines could be used as potential targets for cancer therapy.More interestingly,the chromatin state difference detected between MRC-5 and HL7702 cell lines indicated that the chromatin openness serve key roles in iPSC inducing process.The comparison between three subtype of cervix cancer emphasized the influence of human papillomavirus infection in this cancer which highly threaten women health.3.Decoding genetic and epigenetic information embedded in cell free DNA with adapted SALP-seqCell free DNA(cfDNA)in human plasma carries abundant information,which has therefore been the key sample for non-invasive prenatal testing(NIPT)and liquid biopsy.Especially by using the rapidly developed next-generation sequencing(NGS)techniques,the genetic and epigenetic information embedded in cfDNA has been effectively and extensively decoded.In this process,a key step is to construct the NGS library.Due to its high degradation,the single strand-based method was reported to be more qualified to construct the NGS library of cfDNA.In order to develop a new simple method for this application,this study adapted our recently developed single strand adaptor library preparation(SALP)method for constructing an NGS library of cfDNA.In the improved method,cfDNA was firstly denatured into single strands and then ligated with a single strand adaptor(SSA)that had a 3?overhang of 3 random bases by using T4 DNA ligase.The SSA-ligated DNA was converted into double-stranded DNA with an additional adenine at the other end by polymerizing with Taq polymerase.Next,a barcode T adaptor(BTA)was ligated to this end.Finally,the cfDNA ligated with two adaptors was amplified with the Illumina-compatible primers for NGS.Using the method,this study successfully sequenced 20 cfDNA samples from 16 esophageal cancer patients and 4 healthy people.By bioinformatics analysis,this study found the genetic and epigenetic difference between patients and healthy people and identified 23 epigenetic and 28 genetic altered esophageal cancer-specific genes.4.Binding feature of NF-?B in HeLa cell lineNuclear factor?B(NF-?B)is a DNA-binding transcription factor.Characterizing its genomic binding sites is crucial for understanding its gene regulatory function and mechanism in cells.This study characterized the binding sites of NF-kB RelA/p65 in the tumor neurosis factor-?(TNF?)stimulated HeLa cells by a precise chromatin immunoprecipitation-sequencing(ChIP-seq).The results revealed that NF-?B binds nontraditional motifs(nt-motifs)containing conserved GGAA quadruplet.Moreover,nt-motifs mainly distribute in the peaks nearby centromeres that contain a larger number of repetitive elements such as satellite,simple repeats and short interspersed nuclear elements(SINEs).This intracellular binding pattern was then confirmed by the in vitro detection,indicating that NF-?B dimers can bind the nontraditional?B(nt-?B)sites with low affinity.However,this binding hardly activates transcription.This study thus deduced that NF-?B binding nt-motifs may realize functions other than gene regulation as NF-?B binding traditional motifs(t-motifs).To testify the deduction,many ChIP-seq data of other cell lines were then analyzed.The results indicate that NF-?B binding nt-motifs is also widely present in other cells.The ChIP-seq data analysis also revealed that nt-motifs more widely distribute in the peaks with low-fold enrichment.Importantly,it was also found that NF-?B binding nt-motifs is mainly present in the resting cells,whereas NF-?B binding t-motifs is mainly present in the stimulated cells.Astonishingly,no known function was enriched by the gene annotation of nt-motif peaks.Based on these results,this study proposed that the nt-?B sites that extensively distribute in larger numbers of repeat elements function as a nuclear reservoir of NF-?B.The nuclear NF-?B proteins stored at nt-?B sites in the resting cells may be recruited to the t-?B sites for regulating its target genes upon stimulation.In summary,a novel NGS library construction method–SALP,was developed which could be used to identify chromatin open state in multiple samples with a unbiased way.Using SALP,the chromatin open state of totally 14 type of cells were analyzed,and several comparison were performed in different aspects.With further optimization of SALP,this method could be used to construct cfDNA NGS library.After analyzing the 20 cfDNA samples from esophageal cancer patients and healthy person,the cfDNA sequencing was successfully used in detecting chromatin state.And 23 gene with significant chromatin open state difference between esophageal cancer patients and healthy person were identified,which provided new targets for esophageal cancer therapy and test.The binding feature of NF-?B with highly repetitive sequence in HeLa cell line identified in this research proved new insight for understanding the role of genome repetitive elements in gene transcription regulation.