A long-read sequencing strategy with overlapping linkers on adjacent fragments (OLAF-Seq) for targeted resequencing and enrichment.

In this report, we present OLAF-Seq, a novel strategy to construct a long-read sequencing library such that adjacent fragments are linked with end-terminal duplications. We use the CRISPR-Cas9 nickase enzyme and a pool of multiple sgRNAs to perform non-random fragmentation of targeted long DNA molecules (> 300kb) into smaller library-sized fragments (about 20 kbp) in a manner so as to retain physical linkage information (up to 1000 bp) between adjacent fragments. DNA molecules targeted for fragmentation are preferentially ligated with adaptors for sequencing, so this method can enrich targeted regions while taking advantage of the long-read sequencing platforms. This enables the sequencing of target regions with significantly lower total coverage, and the genome sequence within linker regions provides information for assembly and phasing. We demonstrated the validity and efficacy of the method first using phage and then by sequencing a panel of 100 full-length cancer-related genes (including both exons and introns) in the human genome. When the designed linkers contained heterozygous genetic variants, long haplotypes could be established. This sequencing strategy can be readily applied in both PacBio and Oxford Nanopore platforms for both long and short genes with an easy protocol. This economically viable approach is useful for targeted enrichment of hundreds of target genomic regions and where long no-gap contigs need deep sequencing.

Single-molecule telomere length characterization by optical mapping in nano-channel array: Perspective and review on telomere length measurement.

In humans, the telomere consists of tandem 5'TTAGGG3' DNA repeats on both ends of all 46 chromosomes. Telomere shortening has been linked to aging and age-related diseases. Similarly, telomere length changes have been associated with chemical exposure, molecular-level DNA damage, and tumor development. Telomere elongation has been associated to tumor development, caused due to chemical exposure and molecular-level DNA damage. The methods used to study these effects mostly rely on average telomere length as a biomarker. The mechanisms regulating subtelomere-specific and haplotype-specific telomere lengths in humans remain understudied and poorly understood, primarily because of technical limitations in obtaining these data for all chromosomes. Recent studies have shown that it is the short telomeres that are crucial in preserving chromosome stability. The identity and frequency of specific critically short telomeres potentially is a useful biomarker for studying aging, age-related diseases, and cancer. Here, we will briefly review the role of telomere length, its measurement, and our recent single-molecule telomere length measurement assay. With this assay, one can measure individual telomere lengths as well as identify their physically linked subtelomeric DNA. This assay can also positively detect telomere loss, characterize novel subtelomeric variants, haplotypes, and previously uncharacterized recombined subtelomeres. We will also discuss its applications in aging cells and cancer cells, highlighting the utility of the single molecule telomere length assay.

Customized optical mapping by CRISPR-Cas9 mediated DNA labeling with multiple sgRNAs.

Whole-genome mapping technologies have been developed as a complementary tool to provide scaffolds for genome assembly and structural variation analysis (1,2). We recently introduced a novel DNA labeling strategy based on a CRISPR-Cas9 genome editing system, which can target any 20bp sequences. The labeling strategy is specifically useful in targeting repetitive sequences, and sequences not accessible to other labeling methods. In this report, we present customized mapping strategies that extend the applications of CRISPR-Cas9 DNA labeling. We first design a CRISPR-Cas9 labeling strategy to interrogate and differentiate the single allele differences in NGG protospacer adjacent motifs (PAM sequence). Combined with sequence motif labeling, we can pinpoint the single-base differences in highly conserved sequences. In the second strategy, we design mapping patterns across a genome by selecting sets of specific single-guide RNAs (sgRNAs) for labeling multiple loci of a genomic region or a whole genome. By developing and optimizing a single tube synthesis of multiple sgRNAs, we demonstrate the utility of CRISPR-Cas9 mapping with 162 sgRNAs targeting the 2Mb Haemophilus influenzae chromosome. These CRISPR-Cas9 mapping approaches could be particularly useful for applications in defining long-distance haplotypes and pinpointing the breakpoints in large structural variants in complex genomes and microbial mixtures.

Single-molecule analysis of subtelomeres and telomeres in Alternative Lengthening of Telomeres (ALT) cells.

BACKGROUND: Telomeric DNA is typically comprised of G-rich tandem repeat motifs and maintained by telomerase (Greider CW, Blackburn EH; Cell 51:887-898; 1987). In eukaryotes lacking telomerase, a variety of DNA repair and DNA recombination based pathways for telomere maintenance have evolved in organisms normally dependent upon telomerase for telomere elongation (Webb CJ, Wu Y, Zakian VA; Cold Spring Harb Perspect Biol 5:a012666; 2013); collectively called Alternative Lengthening of Telomeres (ALT) pathways. By measuring (TTAGGG) n tract lengths from the same large DNA molecules that were optically mapped, we simultaneously analyzed telomere length dynamics and subtelomere-linked structural changes at a large number of specific subtelomeric loci in the ALT-positive cell lines U2OS, SK-MEL-2 and Saos-2. RESULTS: Our results revealed loci-specific ALT telomere features. For example, while each subtelomere included examples of single molecules with terminal (TTAGGG) n tracts as well as examples of recombinant telomeric single molecules, the ratio of these molecules was subtelomere-specific, ranging from 33:1 (19p) to 1:25 (19q) in U2OS. The Saos-2 cell line shows a similar percentage of recombinant telomeres. The frequency of recombinant subtelomeres of SK-MEL-2 (11%) is about half that of U2OS and Saos-2 (24 and 19% respectively). Terminal (TTAGGG) n tract lengths and heterogeneity levels, the frequencies of telomere signal-free ends, and the frequency and size of retained internal telomere-like sequences (ITSs) at recombinant telomere fusion junctions all varied according to the specific subtelomere involved in a particular cell line. Very large linear extrachromosomal telomere repeat (ECTR) DNA molecules were found in all three cell lines; these are in principle capable of templating synthesis of new long telomere tracts via break-induced repair (BIR) long-tract DNA synthesis mechanisms and contributing to the very long telomere tract length and heterogeneity characteristic of ALT cells. Many of longest telomere tracts (both end-telomeres and linear ECTRs) displayed punctate CRISPR/Cas9-dependent (TTAGGG) n labeling patterns indicative of interspersion of stretches of non-canonical telomere repeats. CONCLUSION: Identifying individual subtelomeres and characterizing linked telomere (TTAGGG) n tract lengths and structural changes using our new single-molecule methodologies reveals the structural consequences of telomere damage, repair and recombination mechanisms in human ALT cells in unprecedented molecular detail and significant differences in different ALT-positive cell lines.

A micropatterned substrate for on-surface enzymatic labelling of linearized long DNA molecules.

Optical mapping of linearized DNA molecules is a promising new technology for sequence assembly and scaffolding, large structural variant detection, and diagnostics. This is currently achieved either using nanochannel confinement or by stretching single DNA molecules on a solid surface. While the first method necessitates DNA labelling before linearization, the latter allows for modification post-linearization, thereby affording increased process flexibility. Each method is constrained by various physical and chemical limitations. One of the most common techniques for linearization of DNA uses a hydrophobic surface and a receding meniscus, termed molecular combing. Here, we report the development of a microfabricated surface that can not only comb the DNA molecules efficiently but also provides for sequence-specific enzymatic fluorescent DNA labelling. By modifying a glass surface with two contrasting functionalities, such that DNA binds selectively to one of the two regions, we can control DNA extension, which is known to be critical for sequence-recognition by an enzyme. Moreover, the surface modification provides enzymatic access to the DNA backbone, as well as minimizing non-specific fluorescent dye adsorption. These enhancements make the designed surface suitable for large-scale and high-resolution single DNA molecule studies.