Skip to 0 minutes and 1 secondStrand-seq is a method for single-cell DNA template strand sequencing. It was originally developed for tracking DNA strand segregation during cell division, but has many other applications as well. During DNA replication, a complimentary nascent strand of DNA is made from each original template strand. This creates two identical sister chromatids for each chromosome. During mitosis, sister chromatids are divided equally over two daughter cells, ensuring they inherit identical DNA complements. Because the DNA in both daughter cells is identical, it is impossible to see which cell inherited which template strands.
Skip to 0 minutes and 45 secondsHowever, if cells are pulsed with a dose of the thymidine analogue BrdU for exactly one cell division, the BrdU becomes incorporated into the newly-formed DNA strands, making it possible to distinguish the sister chromatids. Upon cell division, each daughter cell will inherit a template 'Watson' strand which does not contain BrdU and one nascent 'Crick' strand with BrdU, while the other sister chromatid will be the exact mirror image.
Skip to 1 minute and 17 secondsIn order to detect inheritance of template DNA strand at the single cell level, single cells are sorted by means of flow cytometry.
Skip to 1 minute and 27 secondsThe DNA is extracted from each cell and then fragmented. These DNA fragments are further processed for sequencing. The BrdU-labelled DNA is removed, leaving only the original template strand behind. Each DNA fragment is then amplified, sequenced, and aligned to the correct location in the genome. Further analysis is performed by a software package called BAIT, which plots the reads on ideograms and determines the strand state for each chromosome. A chromosome may inherit one Watson template strand and one Crick template strand, each from one of the homologues. It is also possible that a chromosome inherits two Watson or two Crick strands. So a cell with 46 chromosomes will look something like this.
Skip to 2 minutes and 20 secondsUsing Strand-seq, we can now determine which template strands were inherited for each chromosome. And by comparing strand inheritance across many cells, we are able to determine if it is random or not. Interestingly, not all chromosomes inherit intact template strands. Instead, they inherit part of the Watson template strand and part of the Crick template strand. Such a pattern indicates the presence of a sister chromatid exchange, or SCE. These are the result of a DNA double strand break, and the number of SCEs is an indicator for genome instability. Another type of rearrangement is the chromosomal inversion, resulting from a broken chromosome that was put back together incorrectly.
Skip to 3 minutes and 7 secondsIf two chromosomes break at the same time, pieces of the different chromosomes are sometimes pasted together wrongly. This results in chromosomal translocation. Strand-seq can also be used to detect chromosome copy number variations across the genome. It is also possible that chromosomes are not evenly divided over the daughter cells, resulting in a cell containing, for example, three copies of chromosome 18 trisomy. Strand-seq is a very powerful tool for detecting not only DNA inheritance patterns, but also different types of chromosomal aberrations. As such, Strand-seq can be used to assess genomic instability at single cell level, resulting in a much higher sensitivity than previously possible.
Animation of single-cell DNA template strand sequencing (Strand-seq)
The single-cell DNA template strand sequencing technique - Strand-seq for short - is a method to identify the original parental DNA template strands in daughter cells following cell division.
Strand-seq is a very powerful tool for not just detecting DNA strand inheritance patterns, but also for assessing genomic instability at the single cell level. This can be done with a much higher sensitivity and level of resolution than was previously possible. This animation explains the principle of Strand-seq and also briefly introduces the main applications of this method.
© University of Groningen / University Medical Center Groningen