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Comparative Study
, 8 (1), 9436

Methodology for Y Chromosome Capture: A Complete Genome Sequence of Y Chromosome Using Flow Cytometry, Laser Microdissection and Magnetic Streptavidin-Beads

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Comparative Study

Methodology for Y Chromosome Capture: A Complete Genome Sequence of Y Chromosome Using Flow Cytometry, Laser Microdissection and Magnetic Streptavidin-Beads

M J Alvarez-Cubero et al. Sci Rep.

Abstract

This study is a comparison of the efficiency of three technologies used for Y chromosome capture and the next-generation sequencing (NGS) technologies applied for determining its whole sequence. Our main findings disclose that streptavidin-biotin magnetic particle-based capture methodology offers better and a deeper sequence coverage for Y chromosome capture, compared to chromosome sorting and microdissection procedures. Moreover, this methodology is less time consuming and the most selective for capturing only Y chromosomal material, in contrast with other methodologies that result in considerable background material from other, non-targeted chromosomes. NGS results compared between two platforms, NextSeq 500 and SOLID 5500xl, produce the same coverage results. This is the first study to explore a methodological comparison of Y chromosome capture and genetic analysis. Our results indicate an improved strategy for Y chromosome research with applications in several scientific fields where this chromosome plays an important role, such as forensics, medical sciences, molecular anthropology and cancer sciences.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Overview of the three technologies used in this study (flow cytometry capture, laser capture microdissection, magnetic streptavidin-bead capture). For all three processes, human lymphocytes were cultured overnight, then phytohaemagglutinin (PHA) and colcemid were added to achieve a high mitotic index and accumulation of cells in metaphase. Later, metaphase chromosomes were extracted from the lymphocytes. (A) For flow cytometry capture, the extracted chromosomes were incubated with a specific biotin Y chromosome probe and stained with streptavidin-PE and DAPI. Y chromosomes were then sorted in a FACSAria flow cytometer. The sorted chromosomes were collected in Eppendorf (Hamburg, Germany) tubes in ddH2O (double-distilled water) for further processing. (B) For laser capture microdissection, individual chromosomes were hybridized with Y chromosome-specific probes conjugated with FITC (green), counterstained with DAPI (blue) and mounted on slides covered by polyethylene membranes. On these slides, they were selected and catapulted by the laser pressure catapulting (LPC) function in a Zeiss PALM MicroBeam IV Laser Microdissector. Y chromosomes were captured within the cap and dissolved in TE buffer. The cap was closed and the sample was spun down by centrifugation. (C) For magnetic streptavidin-bead capture, chromosomes were incubated with a specific biotin Y chromosome probe as in the previous procedure. Dynabeads MyOne streptavidin beads were added to the probe Y chromosome mixture and magnetic separation was performed to capture the Y chromosome on a magnetic rack. Finally, in all cases, physical fragmentation was performed before library prep and sequencing with a Covaris S2 sonicator.
Figure 2
Figure 2
(A) Microfluidic electrophoretic separation of the different methods of Y chromosome capture using a DNA high sensitivity (HS) Bioanalyzer assay. Sample 1: flow cytometry capture, Sample 2: laser capture microdissection, Sample 3: magnetic streptavidin-bead capture. The HS ladder (on left) ranges from 35 base pair (bp) to 7000 bp. All sample peaks appear between the lower and upper marker peaks (35–10380 bp). (B) Bioanalyzer high sensitivity profiles of each capture technique. The protocol for this assay is as follows: The captured DNA (putative Y chromosome) was sonicated to a size between 150 and 500 bp and after the sonication DNA was loaded on the Bioanalyzer assay. The sonication program (see 4.5.1. DNA Shearing) was tested previously to obtain the specific fragment size, which has been verified to be proper for preparing a DNA library.
Figure 3
Figure 3
Representation of reads that map to each chromosome. (A) Flow cytometry capture. (B) Magnetic streptavidin-bead capture and collection methodology. The X-axis shows each analyzed chromosome and Y-axis shows RPKM (reads per kilobase per million mapped reads) representation.
Figure 4
Figure 4
Reads that map to each chromosome. The X-axis shows the different chromosomes and the Y-axis shows the number of reads per chromosome, normalized by the chromosome length. Samtools was used to extract the number of reads that were mapped to each chromosome from bam files.
Figure 5
Figure 5
Fluorescence dot plot of stained chromosomes from the flow cytometer. The chromosomes were stained with DAPI and streptavidin-PE. The position of chromosome Y is gated.
Figure 6
Figure 6
Fluorescence in situ hybridization (FISH) from lymphocytes was analyzed by confocal laser microscopy imaging. Representative chromosomes counterstained with DAPI (blue) and Y chromosome-specific probe conjugated with FITC (fluorescein isothiocyanate, in green) fluorescence images, their superimposition (merged image), and a transmitted light DIC (differential interference contrast) image (gray) are shown.
Figure 7
Figure 7
High Sensitivity DNA Kit electropherograms of during library preparation in both quality control steps: left: after shearing the DNA, and right: at the end of the library preparation. FU = fluorescence unit.

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