Single-cell genomics – methods and protocols
Hou, Y. et al. (2015) Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing. GigaScience 4:37
Compared to other whole genome amplification methods, REPLI-g Single Cell Kits are the best in class for variant calling and are the optimal solution if SNVs and CNVs are of similar importance, as in tumor heterogeneity or cell evolution research.
Korfhage, C. et al. (2015) Parallel WGA and WTA for Comparative Genome and Transcriptome NGS Analysis Using Tiny Cell Numbers. Curr Protoc Mol Biol. 2015 Jul 1
Korfhage, C. et al. (2015) Whole-Transcriptome Amplification of Single Cells for Next-Generation Sequencing. Curr Protoc Mol Biol. 2015 Jul 1
Korfhage, C. et al. (2013) Whole-genome amplification of single-cell genomes for next-generation sequencing. Curr Protoc Mol Biol. 2013 Oct 11
Zhang, C.-Z. et al. (2015) Calibrating genomic and allelic coverage bias in single-cell sequencing, Nat. Comm. 6, 6822
When analyzing bulk samples using whole genome sequencing or whole exome sequencing, the detection of low-frequency (sub-clonal) variants is commonly achieved by increasing sequencing depth. This strategy becomes prohibitively expensive when trying to capture mutations in cells present at 1% or lower fractions. This paper describes a novel method for single cell sequencing which makes accurate variant detection affordable for rare cells from cancer patients, such as CTCs. It involves sequencing many single cells at low sequencing depth. Since most interesting genetic variants are shared among these cells due to their common lineage, a census-based strategy can be used to combine data from multiple single cells to derive a mutational profile of the sample. Using single cell sequencing instead of bulk sample analysis opens a new dimension for comprehensive analysis of rare cells that seed metastases or contribute to drug resistance.
Cancer, neuroscience or stem cell research
Approaches suitable for solid tumors have proven limited for circulating tumor samples due to the low levels of input DNA. Furthermore, whole genome amplification methods are not always compatible with blood collection tube preservatives. In this study, the REPLI-g Single Cell Kit was successfully used for whole genome amplification when combined with a multiplex targeted resequencing approach. The results could stand as proof of concept for real-time monitoring of patient tumors using noninvasive liquid biopsies.
Zhang, C.-Z. et al. (2015) Chromothripsis from DNA damage in micronuclei. Nature, published online 27 May 2015
Chromothripsis is a catastrophic mutational event that results in massive rearrangements of one or more chromosomes. It helps to drive cancer development through oncogene amplification, tumor suppressor loss and DNA damage response impairment. This milestone paper describes a study on individual cells selected based on live cell imaging and subjected to single cell whole genome sequencing. The approach (called “LookSeq”) allowed identification of the first molecular mechanism of chromothripsis. Whole genome amplification (WGA) with highly uniform genome coverage is a prerequisite for successful single cell sequencing. The superior performance of QIAGEN’s REPLI-g Single Cell Kit in WGA was critical for this research.
Yao, X. et al. (2014) Tumor cells are dislodged into the pulmonary vein during lobectomy., J Thorac Cardiovasc Surg.148(6)
This study focused on the contribution of intraoperative tumor shedding to tumor recurrence, using single cell genetic approaches to distinguish between normal and malignant epithelial cells. Whole genome amplification was performed using the REPLI-g Single Cell Kit, followed by amplicon sequencing using the Lung Cancer Panel within the GeneRead DNAseq Targeted Exon Enrichment Panels for target enrichment. After library preparation, barcoded pools were sequenced with paired-end 150 reads using the Illumina MiSeq. Analysis of copy number variation, nested PCR-based mutation analysis of single cells and targeted sequencing were used to identify tumor cells and determine if they were shed into circulation during surgery. The paper shows that single cell genetic approaches together with patient-matched normal and tumor tissues can accurately quantify the number of shed tumor cells.
Wang, Y.. et al. (2014) Clonal evolution in breast cancer revealed by single nucleus genome sequencing, Nature 512
Sequencing studies of breast tumor cohorts have identified many prevalent mutations, but insight into the genomic diversity within tumors is still difficult to attain in bulk tissues. This paper presents a high-coverage method for whole genome and exome single cell sequencing with applications in clonal and mutational evolution (copy number diversity and subclonal mutation analysis). Multiple displacement amplification (MDA) was performed on individual FACS-sorted nuclei using QIAGEN’s REPLI-g technology. The sequence libraries were first sequenced at low coverage depth (1x). Libraries passing quality control were selected for full genome or exome sequencing. The method shows excellent performance, with uniform coverage, low allelic dropout rates and low false positive error rates for point mutations.
Hou, Y. et al. (2012) Single-cell exome sequencing and monoclonal evolution of a JAK2-negative myeloproliferative neoplasm. Cell 148, 873
Jeong, J.H. (2015) Cholinergic neurons in the dorsomedial hypothalamus regulate mouse brown adipose tissue metabolism, Mol Metab 4
RT-PCR analysis revealed the co-expression of muscarinic acetylcholine receptor (M2 mAChR) and tryptophan hydroxylase 2 (Tph2) in single cells collected from mouse brain slices. When combined with other results, these data support the existence of a novel neurochemically specific circuit that modulates thermogenesis and gene expression in interscapular brown adipose tissue.
Lee, D.K., Jeong, J.H., Chun, S.-K., Chua, S. and Jo, Y.-H. (2015) Interplay between glucose and leptin signaling determines the strength of GABAergic synapses at POMC neurons. Nat. Comm. 6, Article: 6618.
This study investigates the interplay between glucose and leptin signaling in glutamatergic proopiomelanocortin (POMC) neurons. Single cell qRT-PCR was used to measure the abundance of transcripts involved in leptin signaling to help build a model that integrates both glucose and leptin signaling pathways in POMC neurons.
Lee, D. et al. (2015) Apelin-13 Enhances Arcuate POMC Neuron Activity via Inhibiting M-Current. PLoS ONE 10(3): e0119457.doi:10.1371/journal.pone.0119457
This paper focuses on how apelin influences POMC neuron activity. Apelin is an endogenous polypeptide ligand for the G protein-coupled receptor APJ, which is involved in the regulation of a various physiological processes. In this study, single cell qRT-PCR was used to monitor changes in the expression of phospholipase C splice variants involved in signaling. These data support a model for the activation of POMC neurons by apelin-13 and suggest a possible alternative therapeutic target against obesity and type 2 diabetes.
Chen, J. et al. (2015) Heterodimerization of Human Orexin Receptor 1 and Kappa Opioid Receptor Promotes Protein Kinase A/cAMP-Response Element Binding Protein Signaling via a Gαs-Mediated Mechanism. Cell. Signal., 27, 1226–1438.
Single cell analysis was applied to identify co-expression of orexin receptor 1 (OX1R) and kappa opioid receptors (KOR) in rat hippocampal neurons. The cytoplasm of primary rat hippocampal neurons was harvested using patch clamp microelectrode pipettes. Single cell RNA was amplified using REPLI-g WTA Single Cell Kit. PCR products were analyzed on an agarose gel and sequenced to confirm identity. Heterodimerization between OX1R and KOR and the effects on signaling transduction mechanisms were explored using bioluminescence and fluorescence resonance energy transfer. The results of the study provide new insights into OX1R and KOR dimerization and its role in depression.
Hou, Y. et al. (2012) Single-cell exome sequencing and monoclonal evolution of a JAK2-negative myeloproliferative neoplasm. Cell 148, 873.
Aneuploidy and genome profiling in blastomere research
Wells, D. et al. (2015) Clinical utilisation of a rapid low-pass whole genome sequencing technique for the diagnosis of aneuploidy in human embryos prior to implantation. J Med Genet. 2014 Aug;51(8)
Thornhill, A.R. et al. (2015) Karyomapping—a comprehensive means of simultaneous monogenic and cytogenetic PGD: comparison with standard approaches in real time for Marfan syndrome, Journal of Assisted Reproduction and Genetics, Volume 32
Xu, J. et al. (2015) Embryo Genome Profiling by Single-Cell Sequencing for Preimplantation Genetic Diagnosis in a β-Thalassemia Family Clinical Chemistry 61:4
Wang, L. et al. (2014) Detection of Chromosomal Aneuploidy in Human Pre-implantation Embryos by Next Generation Sequencing, Biology of Reproduction March 19,2014
Choi, Y.H. et al. (2015) Accuracy of preimplantation genetic diagnosis in equine in vivo-recovered and in vitro-produced blastocysts. Reprod Fertil Dev. 2015 Mar 17. doi: 10.1071/RD14419.
The researchers worked with 10–30 cell biopsies and conducted whole genome amplification (WGA) followed by multiplexed PCR assays designed to detect a variety of heritable equine diseases. With such small amounts of input material, WGA comprises a crucial step in the workflow, since it’s exceedingly difficult to design specific, accurate multiplexed assays with such little DNA. Choosing the right WGA method is crucial – REPLI-g MDA technology outperformed another MDA-based solution in Choi et al.’s experiments: REPLI-g results were accurate in 98.2% of total loci analyzed.
Microbiology, infectious disease research, metagenomics
Eloe-Fadrosh, E.A. et al. (2016) Global metagenomic survey reveals a new bacterial candidate phylum in geothermal springs. Nat. Commun. 7, Article number: 10476 doi:10.1038/ncomms10476
By analyzing metagenomic data from IMG/M combined with single-cell genomics from samples collected from geothermal springs, Eloe-Fadrosh et al. were able to discover and describe a novel bacterial candidate phylum (‘Candidatus Kryptonia’). Starting with a collection of 31955 long assembled contigs (≥ 100kbp) from 4290 metagenomic data sets (IMG/M), 744 contigs with non-redundant SSU rRNA (small-subunti ribosomal RNA) were further selected, aligned and placed on a reference phylogenetic tree. This led finally to the identification of a distinct lineage and near-complete recovery of four distinct genomes. In addition to metagenomics data mining, single cell sampling from four geothermal springs was performed. Single cells from the samples were isolated using FACS sorting, followed by WGA with REPLI-g Single Cell Kit, PCR screening for genomes from single cells matching ‘Ca. Kryptonia’ SSU rRNA sequences and sequenced on Illumina MiSeq. 18 single cell genomes with an average genome completeness of 67% were recovered. The high-quality draft genomes from metagenomic data analysis and single-cell genomics enabled further analyses in bacteria-virus interaction and the detection of a novel fusion between two different CRISPR-Cas types, which represents the first description of a type I-B/type III-A CRISPR-Cas fusion.
Chen, Yi-ran et al. (2016) Novel species and expanded distribution of ellipsoidal multicellular magnetotactic prokaryotes. Environmental Microbiology Reports 8(2), 218-226
The research in this study is on a specific morphotype of multicellular magnetotactic prokaryotes (MMPs), which are groups of 10 to 100 cells of diverse magnetotactic bacteria. Phylogenetic analysis of these MMPs was performed by isolating the specific morphotype (ellipsoidal MMPs) by micromanipulation, followed by WGA using the REPLI-g Single Cell Kit and 16S rRNA gene based sequence analysis. The phylogenetic analysis revealed similarities and differences of species composition of MMPs from various sampling sites, indicating as well a new candidate species.
Nakamura, K. et al. (2016) Culture-independent method for identification of microbial enzyme-encoding genes by activity-based single-cell sequencing using a water-in-oil microdroplet platform, Sci Rep. 2016 Feb 26;6:22259. doi: 10.1038/srep22259
The authors describe an activity-based single-cell sequencing approach enabling the visual evaluation of microbial cells during screening and offering a new targeted approach of specific isolation of microbial cells. Environmental microbes were encapsulated in water-oil microdroplets (containing a fluorogenic substrate for a target enzyme) This enabled the screening for microdroplets that contain cells active for the specific enzyme. The selected microbial cells were recovered and subjected to whole genome amplification using the REPLI-g Single Cell Kit. Successfully amplified single cell genomes were further analyzed by whole genome sequencing.
Mansor, M. et al. (2015) Metabolic diversity and ecological niches of Achromatium populations revealed with single-cell genomic sequencing, Frontiers in Microbiology, Vol. 6
Single cell sequencing is used in this study to uncover the anabolic and metabolic potential of the bacterial genus Achromatium and the genetic heterogeneity of Achromatium populations. Achromatium cells were enriched from sediment samples of a spring, flow-sorted and finally hand-picked using a micromanipulator. Whole genome amplification of the single cells was done with the REPLI-g Single Cell Kit. The WGA-DNA samples containing 16SrRNA gene sequences affiliated with the genus Achromatium were further analysed by whole genome sequencing. Draft genomes with 80% completeness were achieved. The draft genomes provided a basis of further analyses of the metabolic versatility of Achromatium providing new insights for further discussions on the mechanisms of intracellular calcification.
Nair, S. et al. (2014) Single-cell genomics for dissection of complex malaria infections, Genome Res. 2014. 24:1028-1038
The study describes optimization of an accurate single-cell genomics approach for malaria parasites that is applicable to both cultivable and noncultivable malaria species to reveal within-host variation. To determine malarial genotypes, the researchers developed a single-cell approach in which they isolated single infected red blood cells, used whole genome amplification and then genotyped and sequenced the parasites using next-generation sequencing.
Zoll, J. et al. (2015) Direct multiplexed whole genome sequencing of respiratory tract samples reveals full viral genomic information, Journal of Clinical Virology 66 (2015) 6–11
A proof-of-concept for virome determination by NGS. Virome determination is enabled by parallel preamplification of viral DNA and RNA with the REPLI-g Cell WGA & WTA Kit.
Bavelaar, H.H. (2015) Whole genome sequencing of fecal samples as a tool for the diagnosis and genetic characterization of norovirus. J. Clin. Virol. 72, 122.
A new proof of principle study by Bavelaar et al. shows that NGS is equally applicable in the detection and characterization of other norovirus genotypes. The authors performed direct multiplexed whole genome sequencing on fecal samples from patients with gastroenteritis. The study included eight norovirus positive fecal samples (samples 1–8) that had been previously characterized using the Noronet typing tool and fecal samples from two patients known to have gastroenteritis caused by norovirus but with unknown genotypes (samples 9 and 10). Sufficient amounts of RNA were isolated from all samples to perform whole transcriptome sequencing for the detection of RNA viruses using the REPLI-g WTA Single Cell Kit.
Lai Y., et al. (2015) Metagenomic Human Repiratory Air in a Hospital Environment. PLoS ONE 10(10): e0139044. doi:10.1371/journal.pone.0139044