Improving DNA amplification for single-cell genomics

The single-cell DNA sequencing challenge

Deep sequencing of genomes (Whole Genome Sequencing, WGS) is important not only to improve our knowledge in life sciences and evolutionary biology but also to make clinical progresses. The analysis of the genome and its variations at the cell level have major applications: analysis of mutation rates in somatic cells, including copy-number variations (CNVs) and single-nucleotide variations (SNVs), evolution of cancer, recombination in germ cells, preimplantation genetic analysis for embryos or analysis of microbial populations (mini-metagenomics).

Because of the low amount of DNA in a cell, single-cell whole genome sequencing requires whole genome amplification. The 3 methods currently used are degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), multiple displacement amplification (MDA), and multiple annealing and looping-based amplification cycles (MALBAC). However, these methods have limited capability to detect genomic variants and create amplification bias, artefacts and errors (see the overview by Gawad C. et al.).

New methodology for single-cell whole genome amplification

To overcome the limitations of exponential amplification, Xie group has recently developed the Linear Amplification via Transposon Insertion (LIANTI) method.

LIANTI takes advantage of Tn5 transposition and T7 in vitro transcription to linearly amplify genomic DNA fragments from a single human cell.

LIANTI scheme. Genomic DNA from a single cell is randomly fragmented and tagged by LIANTI transposon, followed by DNA polymerase gap extension to convert single-stranded T7 promoter loops into double-stranded T7 promoters on both ends of each fragment. In vitro transcription overnight is performed to linearly amplify the genomic DNA fragments into genomic RNAs, which are capable of self-priming on the 3′ end. After reverse transcription, RNase digestion, and second-strand synthesis, double-stranded LIANTI amplicons tagged with unique molecular barcodes are formed, representing the amplified product of the original genomic DNA from a single cell, and ready for DNA library preparation and next-generation sequencing. From Chongyi C., et al. Science. 356:189–194.

LIANTI exhibits the highest amplification uniformity compared to the other current WGA methods. It allows accurate detection of single-cell micro-CNVs with kilobase resolution). LIANTI method also achieves the highest amplification fidelity for accurate single-cell SNV detection.

LIANTI amplification uniformity and fidelity. (A) coefficient of variation for read depths along the genome as a function of bin sizes from 1 b to 100 Mb, showing amplification noise on all scales for single-cell WGA methods, including DOP-PCR, MALBAC, MDA, and LIANTI. The normalized MALBAC data (dashed line) are shown together with the unnormalized MALBAC data. Only the unnormalized data of the other methods are shown as no substantial improvement by normalization was observed. Poisson curve is the expected coefficient of variation for read depth assuming only Poisson noise. LIANTI exhibits a much improved amplification uniformity over the previous methods on all scales. (B) False-positive rates of SNV detection in a single BJ cell. The error bars were calculated from three different BJ cells. From Chongyi C., et al. Science. 356:189–194.

The high precision of genomic variants detection by LIANTI method would help improved analysis of single-cell DNA sequences, better diagnosis and understanding the evolution of cancer and other diseases.

Based on the recent papers:

Chen C. et al. (2017) Single-cell whole-genome analyses by Linear Amplification via Transposon Insertion (LIANTI). Science. 356:189–194.

Gawad C. et al. (2016) Single-cell genome sequencing: current state of the science. Nat. Rev. Genet. 17(3):175–88.