Biology:LIANTI
Linear Amplification via Transposon Insertion (LIANTI) is a linear whole genome amplification (WGA) method.[1] To analyze or sequence very small amount of DNA, i.e. genomic DNA from a single cell, the picograms of DNA is subject to WGA to amplify at least thousands of times into nanogram scale, before DNA analysis or sequencing can be carried out. Previous WGA methods (DOP-PCR,[2] MDA,[3] MALBAC[4]) use exponential/nonlinear amplification schemes, leading to bias accumulation and error propagation. LIANTI achieved linear amplification of the whole genome for the first time, enabling more uniform and accurate amplification.[1]
Shown in the figure is a simulation to illustrate the advantages of linear amplification over exponential amplification, assuming two DNA fragments A and B with replication yields of 100% and 70% per round, respectively. First, linear amplification is more even than exponential amplification. To achieve ~10,000 fold amplification of fragment A, exponential amplification results in a ratio of 8:1, hampering the accuracy of CNV detection. In contrast, linear amplification exhibits a ratio of 1:0.7, which is much closer to unity. Second, linear amplification is superior to exponential amplification in fidelity. In exponential amplification, errors generated in early cycles of amplification will be propagated permanently, leading to SNV false-positives. In contrast, in linear amplification, the errors would appear randomly at different locations in the amplicons to be easily filtered out.
General scheme
Genomic DNA is randomly fragmented and tagged by Tn5 transposon insertion containing T7 promoter sequence, and the resulting DNA fragments are linearly amplified into RNAs by T7 in vitro transcription. Following reverse transcription and second strand synthesis, double-stranded DNA amplicons are formed representing the linear amplification product of the original genomic DNA, which is suitable for DNA library preparation and sequencing.
Experimental workflow
1. Cell lysis. Single cells are placed into PCR tubes containing lysis buffer by mouth pipetting, flow sorting, micromanipulator, laser dissection or microfluidic devices. Cells are subsequently lysed by Qiagen protease digestion. If the starting material is small amount of genomic DNA instead of single cells, cell lysis step can be skipped.
2. LIANTI transposome. LIANTI transposome is made by mixing equal molar of Tn5 transposase and LIANTI transposon DNA. The sequence of LIANTI transposon DNA is: 5'/Phos/CTGTCTCTTATACACATCTGAACAGAATTTAATACGACTCACTATAGGGAGATGTGTATAAGAGACAG-3' After self annealing, the LIANTI transposon DNA consists of a 19-bp double-stranded region for Tn5 transposase binding and dimerization, and a 30-nt single-stranded loop containing T7 promoter sequence.
3. Tn5 transposition. Genomic DNA from a single cell is randomly fragmented and tagged by LIANTI transposome insertion during transposition reaction.
4. Gap filling. After Tn5 transposition, both ends of each DNA fragment are gap filled and extended by DNA polymerase extension, converting single-stranded loops into double-stranded T7 promoters on both ends of each fragment. The residue Tn5 transposase and DNA polymerase are subsequently removed by protease digestion, followed by heat inactivation of the protease.
5. In vitro transcription (IVT) linear amplification. Still within the same PCR tube, overnight IVT reaction is assembled, including standard IVT buffer, NTPs, T7 RNA polymerase, RNase inhibitor, DMSO, etc.
6. Reverse transcription (RT). After overnight IVT, RNAs representing the linearly amplified products of the original genomic DNA are column purified, self primed on the 3′ end, and reverse transcribed. RNase digestion is carried out to convert the double-stranded DNA-RNA hybrids into single-stranded DNA.
7. Second strand synthesis (SSS). Taking advantage of the 19-bp specific sequence on the 3' end of each single-stranded DNA, SSS step is performed by specific priming and DNA polymerase extension. The resulting double-stranded DNA fragments are LIANTI amplicons linearly amplified from the original single-cell genomic DNA, with unique molecular barcodes attached on each amplicon.
8. Library prep and sequencing. Depending on transposome insertion density and specific applications, LIANTI amplicons can be subject to optional sonication, before proceeding to standard library prep pipelines (i.e. NEBNext Ultra II DNA Library Prep Kit for Illumina).
Advantages
1. Accurate detection of single-cell copy-number variations (CNVs) with kilobase resolution[1]
LIANTI exhibits the highest amplification uniformity compared to other single-cell WGA methods, as shown by read depth plots across the genome,[1] as well as coefficient of variation for read depths along the genome as a function of bin size.[1] Together with its unique capability of digital counting to infer fragment numbers based on the mapping coordinates of each amplicon,[1] LIANTI allows accurate detection of single-cell micro-CNVs with kilobase resolution. For comparison, other single-cell WGA methods including DOP-PCR, MDA and MALBAC can only detect CNVs with megabase resolution due to amplification noise, thus completely missing micro-CNVs in single cells.[5]
2. Accurate detection of single-cell single-nucleotide variations (SNVs)
By virtue of linear amplification, LIANTI achieves the highest amplification fidelity for single-cell single-nucleotide variation (SNV) detection, even with the low fidelity of T7 RNA polymerase. However, the SNV false positive rate of LIANTI is actually dominated by C-to-T false positives associated with the experimental artifact of cytosine deamination into uracil upon cell lysis.[1] This was evidenced by the SNV false positive spectra of LIANTI and MDA, which are different from that of the bulk. Moreover, uracil-DNA glycosylase (UDG) treatment before IVT linear amplification can eliminate C-to-T false positives by removing uracil generated upon cell lysis. With UDG treatment, LIANTI has a false positive rate (FPR) of 1.7 × 10−6 under a particular experimental condition for single BJ cells.[1]
3. High coverage of the single-cell genome
LIANTI achieves a high genome coverage, typically between 90% and 98%, as well as a low allele dropout rate (ADO), typically around 20%.[1]
Limitations
Even with LIANTI assay combined with UDG treatment to remove C-to-T false positives due to cytosine deamination into uracil upon cell lysis, the remaining false positives (1.7 × 10−6) still prevent accurate detection of SNVs in single cells. Among the remaining false positives, A-to-G is likely caused by adenine deamination,[1][6] and G-to-T is due to guanine oxidation to 8-hydroxyguanine.[1][7][8] As a result, the chemical instability of DNA bases in the absence of cellular DNA repair system upon cell lysis before WGA fundamentally limits the accuracy of single-cell SNV detection. Therefore, sequencing a pair of kindred cells is still necessary to completely rule out SNV false positives in order to call de novo single-cell SNVs.[1][4]
Applications
1. LIANTI has been used to probe stochastic firing of DNA replication origins in single cells.[1] LIANTI was performed in single human fibroblast cells in early S phase, enabling direct observation of DNA replication origin firing and replicon formation based on the detection of single-cell copy-number gain with kilobase resolution. Certain level of cell-to-cell heterogeneity was observed, suggesting a large degree of stochasticity in replication origin firing in early S phase.[1]
2. LIANTI has been used to characterize UV-induced SNVs in single cells.[1] By sequencing kindred cells amplified with LIANTI, the nonrandom spectra and genome-wide distributions of UV-induced SNVs in single human cells were determined.[1] A depletion of mutations was observed in transcribed regions where DNA damage repair is expected to be more efficient.
3. In addition, the high precision of micro-CNV detection and the ability to call individual SNVs in single cells will allow better genetic screening in reproductive medicine and provide valuable information about how genome variation takes place in cancer and other diseases.
External links
LIANTI paper:
LIANTI single-cell DNA sample whole genome sequencing data:
Software package for LIANTI data analysis:
References
- ↑ 1.00 1.01 1.02 1.03 1.04 1.05 1.06 1.07 1.08 1.09 1.10 1.11 1.12 1.13 1.14 1.15 1.16 Chen, C., Xing, D., Tan, L., Li, H., Zhou, G., Huang, L., and Xie, X.S. (2017). "Single-cell whole-genome analyses by Linear Amplification via Transposon Insertion (LIANTI)". Science 356, 189–194.
- ↑ Telenius, H., Carter, N.P., Bebb, C.E., Nordenskjold, M., Ponder, B.A., and Tunnacliffe, A. (1992). "Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer". Genomics 13, 718–725.
- ↑ Dean, F.B., Hosono, S., Fang, L., Wu, X., Faruqi, A.F., Bray-Ward, P., Sun, Z., Zong, Q., Du, Y., Du, J., et al. (2002). "Comprehensive human genome amplification using multiple displacement amplification". Proceedings of the National Academy of Sciences of the United States of America 99, 5261-5266.
- ↑ 4.0 4.1 Zong, C., Lu, S., Chapman, A.R., and Xie, X.S. (2012). "Genome-wide detection of single-nucleotide and copy-number variations of a single human cell". Science 338, 1622–1626.
- ↑ Huang, L., Ma, F., Chapman, A., Lu, S., and Xie, X.S. (2015). "Single-Cell Whole-Genome Amplification and Sequencing: Methodology and Applications". Annual Review of Genomics and Human Genetics 16, 79–102.
- ↑ Tom Strachan, J.G., Patrick Chinnery (2014). Genetics and Genomics in Medicine.
- ↑ Cheng, K.C., Cahill, D.S., Kasai, H., Nishimura, S., and Loeb, L.A. (1992). "8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G----T and A----C substitutions". The Journal of Biological Chemistry 267, 166–172.
- ↑ Chen, L., Liu, P., Evans, T.C., Jr., and Ettwiller, L.M. (2017). "DNA damage is a pervasive cause of sequencing errors, directly confounding variant identification". Science 355, 752–756.
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