Page 187 - Williams Hematology ( PDFDrive )

162 Part IV: Molecular and Cellular Hematology Chapter 11: Genomics 163

global transcription factor binding genome-wide without the limitations 5-mC prior to library preparation and sequencing, which may allow for
of ChIP-seq described above. NGS-based protocols used to determine genome-wide methylation studies at base pair resolution using smaller
chromatin accessibility differ in the approach to the DNA fragmentation amounts of input DNA than WGBS. A new transposase-based tag-
54
step. Three commonly used protocols are DNase-seq, MNase-seq, and mentation method, similar to the approach used for ATAC-seq, also
ATAC-seq. DNase-seq uses DNase I to fragment DNA based on DNase allows for WGBS with very small amounts of input DNA. 55
45
I hypersensitive sites as a marker of chromatin accessibility. MNase-
seq uses micrococcal nuclease (MNase) to cleave the DNA at accessible APPROACHES TO DNA SEQUENCING FOR
46
sites. ATAC-seq uses the hyperactive Tn5 transposase to simultane-
ously fragment (with minimal sequence bias) and add sequencing RESEARCH PURPOSES
47
adaptors to accessible DNA. Another approach to studying chromatin The study of genomics for research purposes has also shifted as a result
accessibility is known as FAIRE-seq, which involves formalin crosslink- of NGS technology. Prior to the broad availability of NGS platforms,
48
ing of DNA to proteins prior to random fragmentation via sonication. most genomics research studies were genome-wide association stud-
A variation of this protocol, called chromosome conformational capture ies (GWASs) that used a microarray platform to assay for significant
(or “3C”), in which chromatin domains are crosslinked, sequenced, and changes in allele frequency from the panel of single nucleotide poly-
analyzed to determine higher-order structural associations, can provide morphisms (SNPs) included on the array (modern arrays often have
details into the spatial organization of a genome. 49 probes to detect the genotype of more than 1 million SNPs). GWASs
56
require large numbers of samples (cases and controls), and are pow-
Next-Generation Sequencing-Based Studies of ered to identify SNPs that are in linkage disequilibrium with an asso-
Chemical Modifications to DNA: DNA Methylation and ciated condition. It is unlikely that the true pathologic variant will be
57
Hydroxy-methylation discovered via a GWAS. Instead, the results of a GWAS could provide
Unless otherwise specified, DNA methylation is generally synony- the basis for a targeted sequencing study to determine the pathologic
mous with cytosine methylation. Cytosine can undergo methylation alteration(s). In the era of decreasing cost and broad availability of NGS,
or hydroxymethylation at its C5 position to form 5-methylcytosine most genomics studies have shifted to a more inclusive discovery plat-
(5-mC) or 5-hydroxymethylcytosine (5-hmC). Both cytosine methy- form, such as whole-genome sequencing or exome sequencing. Using
lation and 5-hydroxymethylation typically occur when a 5′ cytosine is a platform with single-base resolution rather than a defined content
positioned directly adjacent to a downstream guanine (known as a CpG microarray increases the power to identify a pathologic variant, and the
dinucleotide). There are approximately 26 million CpGs in the human number of samples may decrease. However, for complex genetic dis-
genome. The first genome-wide platforms to detect DNA methylation eases, in which multiple genes may play a causative role, the number of
changes at base pair resolution were microarrays designed to hybrid- samples required remains large and can be cost prohibitive. In these sit-
ize targeted CpGs across the genome (current methylation microarrays uations, investigators often use a combination of GWAS methods (with
target approximately 500,000 CpGs). However, the design of CpG rep- cheaper microarrays) to perform the initial discovery work followed by
resentation on a microarray was often biased toward gene promoters or region-specific NGS discovery sequencing.
other areas of predetermined interest. Several ethical issues complicate the NGS-based study of human
Many protocols exist for differential fragmentation of a genome genomes. First, sequencing data may be potentially “identifiable,” mean-
based on DNA methylation prior to array capture. For example, meth- ing that one could potentially determine another person’s identity based
ylated cytosines are protected from cleavage by particular restriction on sequencing results obtained by a genomic study, when compared to
enzymes: HpaII will cleave C-C-G-G but not C-5mC-G-G, whereas data from a second genotyping assay (such as for diagnostic or criminal
MspI will cleave both sites. By creating separate fragmentation librar- purposes). The Genetic Information Nondiscrimination Act (GINA)
ies using each individual enzyme and then hybridizing each library to of 2008 made it illegal in the United States for employers and health
a separate array, differentially methylated sites can be determined. insurance providers to discriminate based on the results of genetic find-
50
Alternatively, one can perform DNA methylation studies using the ings. However, persons enrolling into genomics research trials must be
sodium bisulfite conversion of cytosine to uracil (which is read as a thy- informed of this theoretical risk of identifiability and be properly con-
midine). Both 5-mC and 5-hmC do not undergo bisulfite conversion sented. Another consequence of genomics studies is that researchers
and are read out as cytosine in a downstream assay. Microarrays that must consider the return of genetic results to patients. The return of
were designed for bisulfite-treated DNA have distinct paired probe sets results is divided into two general categories: incidental findings and
that are designed to capture specific differentially methylated CpGs. findings pertinent to the condition being studied. There is no standard
NGS has enabled the direct sequencing of bisulfite converted DNA for approach for return of results as the approach varies on a case-by-case
unbiased evaluation of methylation and hydroxymethylation genome- basis, depending on the sequencing study and the result to be commu-
51
58
wide. In whole-genome bisulfite sequencing (WGBS), a standard nicated; however, new guidelines are emerging. There is general con-
sequencing library is prepared with methylated C–containing adaptors, sensus in the genomics research community that these issues, and how
followed by the bisulfite conversion of the library. WGBS is complicated they will be handled for the particular study, must be clearly presented
by numerous factors, including (1) the large amount of input DNA in the study protocol and the informed consent documentation.
necessary for sequencing (bisulfite conversion results in DNA degra- The sequencing of cancer genomes, whether by whole-genome
dation), (2) incomplete conversion of cytosine to uracil, and (3) the sequencing, exome sequencing, or multigene panels, is also associated
analytic challenge of determining accurately which sequencing reads with several specific considerations. Proper informed consent is again
have been converted because of the presence of cytosine methylation paramount. Proper sample banking is critical to avoid degradation
or hydroxymethylation. To determine if cytosines are methylated ver- of nucleic acids prior to their isolation, as high-quality DNA or RNA
sus hydroxymethylated, researchers have designed alternative proto- increases the likelihood of successful sequencing independent of the
cols with an added chemical or enzyme-mediated conversion step or NGS platform used. For DNA sequencing studies of a cancer sample, a
antibody-mediated differential capture of 5-mC and 5-hmC prior to matched “normal” sample is often also sequenced to discern the somatic
sequencing. 52,53 Capture-based methods can also be used to target only versus germline status of any identified alterations.

Kaushansky_chapter 11_p0155-0164.indd 162 9/18/15 11:48 PM