P-533 Single-cell long-read nanopore sequencing as a fast and cost-efficient method for aneuploidy detection and potentially PGT-A, a pre-clinical study
Abstract Study question Is long-read nanopore sequencing feasible and reproducible as routine technique for aneuploidy detection, potentially allowing fresh embryo transfer PGT-A cycles? Summary answer Pre-clinical analysis of euploid and aneuploid DNA and single cells using long-read nanopore seque...
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Veröffentlicht in: | Human reproduction (Oxford) 2022-06, Vol.37 (Supplement_1) |
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Sprache: | eng |
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Zusammenfassung: | Abstract
Study question
Is long-read nanopore sequencing feasible and reproducible as routine technique for aneuploidy detection, potentially allowing fresh embryo transfer PGT-A cycles?
Summary answer
Pre-clinical analysis of euploid and aneuploid DNA and single cells using long-read nanopore sequencing resulted in high concordance and reproducible results for aneuploidy detection.
What is known already
PGT-A is mostly performed using short-read next-generation sequencing, which requires high initial investment costs and high running expenses. Third-generation sequencing is a novel sequencing technology with the potential of fast, easy, and cost-effective sequencing analysis, possible even for small and less well-financed clinics. Long-read nanopore sequencing for PGT was mainly shown for structural variants and monogenetic disease, which comes with high costs and is far from clinical routine. PGT-A from trophectoderm biopsy samples using nanopore sequencing was so far demonstrated in a small pilot study and further pre-clinical and clinical studies are needed to transfer the technology into clinical routine.
Study design, size, duration
In this pre-clinical study, euploid and aneuploid DNA and single cells, as well as three to 20 cells were analyzed for aneuploidy using two different whole genome amplification (WGA) kits and long-read nanopore sequencing. In total, 44 different samples were analyzed after multiple displacement amplification (MDA) using REPLIg WGA kit (QIAGEN) and so far, 15 samples were analyzed using PicoPlex WGA kit (Takara) from April 2021. To confirm reproducibility, certain WGA samples were sequenced repetitively.
Participants/materials, setting, methods
Different euploid and aneuploid gDNA samples were diluted to 4.5 pg DNA per sample. Four different human fibroblast cell lines were diluted to approx. 20 cells, or single cells/three cells were picked using micromanipulation technique. DNA and cells were amplified, prepared for sequencing, and sequenced on MinION sequencer from Oxford Nanopore Technology. Data were analyzed using a custom pipeline consisting of pre-processing, alignment and copy-number calling. QC values and whole chromosome aneuploidies were determined automatically.
Main results and the role of chance
From 44 different single cells (n = 14), few cells (n = 9) or diluted gDNA (n = 21) samples amplified using MDA, 42 samples showed good quality sequencing results. Two samples result in QC failure, yielding a sample- |
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ISSN: | 0268-1161 1460-2350 |
DOI: | 10.1093/humrep/deac107.491 |