2 research outputs found
Microfluidic Whole Genome Amplification Device for Single Cell Sequencing
We
developed a microfluidic device to perform multiplex single-cell
whole-genome amplification (WGA) using multiple annealing and looping-based
amplification cycles (MALBAC). This device, made of polydimethylsiloxane
(PDMS), allows us to monitor the whole process of cell loading and
single-cell WGA for sequencing. We show that the genome coverage of
MALBAC amplifications is reproducible between chambers on a single
chip and between different chips, which enables data normalization
using standard samples to accurately identify copy number variations
(CNVs). This device provides an easy-to-operate approach to perform
single cell sequencing library preparation with minimum hands-on time.
It reduces the requirement of manual expertise as well as the risk
of contamination, which is essential in future applications especially
the medical diagnosis
Single-Cell-Based Platform for Copy Number Variation Profiling through Digital Counting of Amplified Genomic DNA Fragments
We
develop a novel single-cell-based platform through digital counting
of amplified genomic DNA fragments, named multifraction amplification
(mfA), to detect the copy number variations (CNVs) in a single cell.
Amplification is required to acquire genomic information from a single
cell, while introducing unavoidable bias. Unlike prevalent methods
that directly infer CNV profiles from the pattern of sequencing depth,
our mfA platform denatures and separates the DNA molecules from a
single cell into multiple fractions of a reaction mix before amplification.
By examining the sequencing result of each fraction for a specific
fragment and applying a segment-merge maximum likelihood algorithm
to the calculation of copy number, we digitize the sequencing-depth-based
CNV identification and thus provide a method that is less sensitive
to the amplification bias. In this paper, we demonstrate a mfA platform
through multiple displacement amplification (MDA) chemistry. When
performing the mfA platform, the noise of MDA is reduced; therefore,
the resolution of single-cell CNV identification can be improved to
100 kb. We can also determine the genomic region free of allelic drop-out
with mfA platform, which is impossible for conventional single-cell
amplification methods