Single-cell DNA template strand sequencing, or Strand-seq, is a technique for the selective sequencing of a daughter cell's parental template strands. This technique offers a wide variety of applications, including the identification of sister chromatid exchanges in the parental cell prior to segregation, the assessment of non-random segregation of sister chromatids, the identification of misoriented contigs in genome assemblies, de novo genome assembly of both haplotypes in diploid organisms including humans, whole-chromosome haplotyping, and the identification of germline and somatic genomic structural variation, the latter of which can be detected robustly even in single cells.
Background Strand-seq (single-cell and single-strand sequencing) was one of the first single-cell sequencing protocols described in 2012. This genomic technique selectively sequencings the parental template strands in single daughter cells DNA libraries. As a proof of concept study, the authors demonstrated the ability to acquire sequence information from the Watson and/or Crick chromosomal strands in an individual DNA library, depending on the mode of chromatid segregation; a typical DNA library will always contain DNA from both strands. The authors were specifically interested in showing the utility of strand-seq in detecting sister chromatid exchanges (SCEs) at high-resolution. They successfully identified eight putative SCEs in the murine (mouse) embryonic stem (meS) cell line with resolution up to 23 bp. This methodology has also been shown to hold great utility in discerning patterns of non-random chromatid segregation, especially in stem cell lineages. Furthermore, SCEs have been implicated as diagnostic indicators of genome stress, information that has utility in cancer biology. Most research on this topic involves observing the assortment of chromosomal template strands through many cell development cycles and correlating non-random assortment with particular cell fates. Single-cell sequencing protocols were foundational in the development of this technique, but they differ in several aspects.
Methodology
Similar methods Past methods have been used to track the inheritance patterns of chromatids on a per-strand basis and elucidate the process of non-random segregation:
Pulse-chase Pulse-chase experiments have been used for determining the segregation patterns of chromosomes in addition to studying other time-dependent cellular processes. Briefly, pulse-chase assays allow researchers to track radioactively labelled molecules in the cell. In experiments used to study non-random chromosome assortment, stem cells are labeled or "pulsed" with a nucleotide analog that is incorporated in the replicated DNA strands. This allows the nascent stands to be tracked through many rounds of replication. Unfortunately, this method is found to have poor resolution as it can only be observed at the chromatid level.
Chromosome-orientation fluorescence in situ hybridization (CO-FISH) CO-FISH, or strand-specific fluorescence in situ hybridization, facilitates strand-specific targeting of DNA with fluorescently-tagged probes. It exploits the uniform orientation of major satellites relative to the direction of telomeres, thus allowing strands to be unambiguously designated as "Watson" or "Crick" strands. Using unidirectional probes that recognize major satellite regions, coupled to fluorescently labelled dyes, individual strands can be bound. To ensure that only the template strand is labelled, the newly formed strands must be degraded by BrdU incorporation and photolysis. This protocol offers improved cytogenetic resolution, allowing researchers to observe single strands as opposed to whole chromatids with pulse-chase experiments. Moreover, non-random segregation of chromatids can be directly assayed by targeting major satellite markers.
Wet lab protocols Cells of interest are cultured either in vivo or in vitro. During S-phase cells are treated with bromodeoxyuridine (BrdU) which is then incorporated into their nascent DNA, acting as a substitute for thymidine. After at least one replication event has occurred, the daughter cells are synchronized at the G2 phase and individually separated by fluorescence-activated cell sorting (FACS). The cells are directly sorted into lysis buffer and their DNA is extracted. Having been arrested at a specified number of generations (usually one), the inheritance patterns of sister chromatids can be assessed. The following methods concentrate on the DNA sequencing of a single daughter cell's DNA. At this point the chromosomes are composed of nascent strands with BrdU in place of thymidine and the original template strands are primed for DNA sequencing library preparation. Since this protocol was published in 2012, the canonical methodology is only well described for Illumina sequencing platforms; the protocol could very easily be adapted for other sequencing platforms, depending on the application. Next, the DNA is incubated with a special dye such that when the BrdU-dye complex is excited by UV light, nascent strands are nicked by photolysis. This process inhibits polymerase chain reaction (PCR) amplification of the nascent strand, allowing only the parental template strands to be amplified. Library construction proceeds as normal for Illumina paired-end sequencing. Multiplexing PCR primers are then ligated to the PCR amplicons with hexamer barcodes identifying which cell each fragment they are derived from. Unlike single cell sequencing protocols, Strand-seq does not utilize multiple displacement amplification or MALBAC for DNA amplification. Rather, it is solely dependent on PCR.
Bioinformatic processing
… excerpt ends here. Continue reading the full article.
