+ Site Statistics
+ Search Articles
+ PDF Full Text Service
How our service works
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ Translate
+ Recently Requested

Whole-genome amplification of single-cell genomes for next-generation sequencing



Whole-genome amplification of single-cell genomes for next-generation sequencing



Current Protocols in Molecular Biology 104: 7.14.1



DNA sequence analysis and genotyping of biological samples using next-generation sequencing (NGS), microarrays, or real-time PCR is often limited by the small amount of sample available. A single cell contains only one to four copies of the genomic DNA, depending on the organism (haploid or diploid organism) and the cell-cycle phase. The DNA content of a single cell ranges from a few femtograms in bacteria to picograms in mammalia. In contrast, a deep analysis of the genome currently requires a few hundred nanograms up to micrograms of genomic DNA for library formation necessary for NGS sequencing or labeling protocols (e.g., microarrays). Consequently, accurate whole-genome amplification (WGA) of single-cell DNA is required for reliable genetic analysis (e.g., NGS) and is particularly important when genomic DNA is limited. The use of single-cell WGA has enabled the analysis of genomic heterogeneity of individual cells (e.g., somatic genomic variation in tumor cells). This unit describes how the genome of single cells can be used for WGA for further genomic studies, such as NGS. Recommendations for isolation of single cells are given and common sources of errors are discussed.

Please choose payment method:






(PDF emailed within 0-6 h: $19.90)

Accession: 056942339

Download citation: RISBibTeXText

PMID: 24510298

DOI: 10.1002/0471142727.mb0714s104


Related references

Single primer isothermal amplification (SPIA) combined with next generation sequencing provides complete bovine coronavirus genome coverage and higher sequence depth compared to sequence-independent single primer amplification (SISPA). Plos one 12(11): E0187780, 2017

Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells. Oncotarget (): -, 2016

Next-generation Sequencing (NGS) Analysis on Single Circulating Tumor Cells (CTCs) with No Need of Whole-genome Amplification (WGA). Cancer Genomics and Proteomics 14(3): 173-179, 2017

Single-Cell Whole-Genome Amplification and Sequencing: Methodology and Applications. Annual Review of Genomics and Human Genetics 16: 79-102, 2015

Microfluidic whole genome amplification device for single cell sequencing. Analytical Chemistry 86(19): 9386-9390, 2014

Identification of optimum sequencing depth especially for de novo genome assembly of small genomes using next generation sequencing data. Plos one 8(4): E60204, 2013

Universal amplification, next-generation sequencing, and assembly of HIV-1 genomes. Journal of Clinical Microbiology 50(12): 3838-3844, 2012

Comparison of whole genome amplification techniques for human single cell exome sequencing. Plos one 12(2): E0171566, 2017

Uniform and accurate single-cell sequencing based on emulsion whole-genome amplification. Proceedings of the National Academy of Sciences of the United States of America 112(38): 11923, 2015

Comprehensive Genome Profiling of Single Sperm Cells by Multiple Annealing and Looping-Based Amplification Cycles and Next-Generation Sequencing from Carriers of Robertsonian Translocation. Annals of Human Genetics 81(2): 91-97, 2017

A18 Random amplification with next-generation sequencing to cover HIV and HCV full-length genomes. Virus Evolution 3(Suppl 1):, 2017

Identification and complete genome sequencing of paramyxoviruses in mallard ducks (Anas platyrhynchos) using random access amplification and next generation sequencing technologies. Virology Journal 8: 463, 2011

Sequence-Independent, Single-Primer Amplification Next-Generation Sequencing of Hantaan Virus Cell Culture-Based Isolates. American Journal of Tropical Medicine and Hygiene 96(2): 389-394, 2017

Systematic evaluation of signal-to-noise ratio in variant detection from single cell genome multiple displacement amplification and exome sequencing. Bmc Genomics 19(1): 681, 2018

Heat-Induced Fragmentation and Adapter-Assisted Whole Genome Amplification Using GenomePlex® Single-Cell Whole Genome Amplification Kit (WGA4). Methods in Molecular Biology 1347: 101-109, 2015