Systematic evaluation of signal-to-noise ratio in variant detection from single cell genome multiple displacement amplification and exome sequencing
Abstract Background The current literature on single cell genomic analyses on the DNA level is conflicting regarding requirements for cell quality, amplification success rates, allelic dropouts and resolution, lacking a systematic comparison of multiple cell input down to the single cell. We hypothe...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2018-09-01
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| Series: | BMC Genomics |
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| Online Access: | http://link.springer.com/article/10.1186/s12864-018-5063-5 |
| _version_ | 1818558423819616256 |
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| author | Anita T. Simonsen Marcus C. Hansen Eigil Kjeldsen Peter L. Møller Johnny J. Hindkjær Peter Hokland Anni Aggerholm |
| author_facet | Anita T. Simonsen Marcus C. Hansen Eigil Kjeldsen Peter L. Møller Johnny J. Hindkjær Peter Hokland Anni Aggerholm |
| author_sort | Anita T. Simonsen |
| collection | DOAJ |
| description | Abstract Background The current literature on single cell genomic analyses on the DNA level is conflicting regarding requirements for cell quality, amplification success rates, allelic dropouts and resolution, lacking a systematic comparison of multiple cell input down to the single cell. We hypothesized that such a correlation assay would provide an approach to address the latter issues, utilizing the leukemic cell line OCI-AML3 with a known set of genetic aberrations. Results By analyzing single and multiple cell replicates (2 to 50 cells) purified by micromanipulation and serial dilution we stringently assessed the signal-to-noise ratio (SNR) from single as well as a discrete number of cells based on a multiple displacement amplification method, with whole exome sequencing as signal readout. In this setting, known OCI-AML3 mutations as well as large copy number alterations could be identified, adding to the current knowledge of cytogenetic status. The presence of DNMT3A R882C, NPM1 W288 fs and NRAS Q61L was consistent, in spite of uneven allelic read depths. In contrast, at the level of single cells, we observed that one-third to half of all variants were not reproduced in the replicate sample, and this allelic mismatch displayed an exponential function of cell input. Large signature duplications were discernible from 5 cells, whereas deletions were visible down to the single cell. Thus, even under highly optimized conditions, single cell whole genome amplification and interpretation must be taken with considerable caution, given that allelic change is frequent and displays low SNR. Allelic noise is rapidly alleviated with increased cell input, and the SNR is doubled from 2 to 50 cells. Conclusions In conclusion, we demonstrate noisy allele distributions, when analyzing genetic aberrations within single cells relative to multiple cells. Based on the presented data we recommend that single cell analyses should include replicate cell dilution assays for a given setup for relative assessment of procedure-specific SNR to ensure that the resolution supports the specific hypotheses. |
| first_indexed | 2024-12-14T00:12:16Z |
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| id | doaj.art-f9324b4dc0d94ba3be4b888025d15cfc |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-12-14T00:12:16Z |
| publishDate | 2018-09-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-f9324b4dc0d94ba3be4b888025d15cfc2022-12-21T23:25:42ZengBMCBMC Genomics1471-21642018-09-011911910.1186/s12864-018-5063-5Systematic evaluation of signal-to-noise ratio in variant detection from single cell genome multiple displacement amplification and exome sequencingAnita T. Simonsen0Marcus C. Hansen1Eigil Kjeldsen2Peter L. Møller3Johnny J. Hindkjær4Peter Hokland5Anni Aggerholm6Department of Hematology, Aarhus University HospitalDepartment of Hematology, Aarhus University HospitalDepartment of Hematology, Aarhus University HospitalDepartment of Hematology, Aarhus University HospitalAAGAARD Skejby Fertility ClinicDepartment of Hematology, Aarhus University HospitalDepartment of Hematology, Aarhus University HospitalAbstract Background The current literature on single cell genomic analyses on the DNA level is conflicting regarding requirements for cell quality, amplification success rates, allelic dropouts and resolution, lacking a systematic comparison of multiple cell input down to the single cell. We hypothesized that such a correlation assay would provide an approach to address the latter issues, utilizing the leukemic cell line OCI-AML3 with a known set of genetic aberrations. Results By analyzing single and multiple cell replicates (2 to 50 cells) purified by micromanipulation and serial dilution we stringently assessed the signal-to-noise ratio (SNR) from single as well as a discrete number of cells based on a multiple displacement amplification method, with whole exome sequencing as signal readout. In this setting, known OCI-AML3 mutations as well as large copy number alterations could be identified, adding to the current knowledge of cytogenetic status. The presence of DNMT3A R882C, NPM1 W288 fs and NRAS Q61L was consistent, in spite of uneven allelic read depths. In contrast, at the level of single cells, we observed that one-third to half of all variants were not reproduced in the replicate sample, and this allelic mismatch displayed an exponential function of cell input. Large signature duplications were discernible from 5 cells, whereas deletions were visible down to the single cell. Thus, even under highly optimized conditions, single cell whole genome amplification and interpretation must be taken with considerable caution, given that allelic change is frequent and displays low SNR. Allelic noise is rapidly alleviated with increased cell input, and the SNR is doubled from 2 to 50 cells. Conclusions In conclusion, we demonstrate noisy allele distributions, when analyzing genetic aberrations within single cells relative to multiple cells. Based on the presented data we recommend that single cell analyses should include replicate cell dilution assays for a given setup for relative assessment of procedure-specific SNR to ensure that the resolution supports the specific hypotheses.http://link.springer.com/article/10.1186/s12864-018-5063-5Single cell sequencingSparse cell sequencingAllele dropoutSignal-to-noise ratioWhole exome sequencingNGS |
| spellingShingle | Anita T. Simonsen Marcus C. Hansen Eigil Kjeldsen Peter L. Møller Johnny J. Hindkjær Peter Hokland Anni Aggerholm Systematic evaluation of signal-to-noise ratio in variant detection from single cell genome multiple displacement amplification and exome sequencing BMC Genomics Single cell sequencing Sparse cell sequencing Allele dropout Signal-to-noise ratio Whole exome sequencing NGS |
| title | Systematic evaluation of signal-to-noise ratio in variant detection from single cell genome multiple displacement amplification and exome sequencing |
| title_full | Systematic evaluation of signal-to-noise ratio in variant detection from single cell genome multiple displacement amplification and exome sequencing |
| title_fullStr | Systematic evaluation of signal-to-noise ratio in variant detection from single cell genome multiple displacement amplification and exome sequencing |
| title_full_unstemmed | Systematic evaluation of signal-to-noise ratio in variant detection from single cell genome multiple displacement amplification and exome sequencing |
| title_short | Systematic evaluation of signal-to-noise ratio in variant detection from single cell genome multiple displacement amplification and exome sequencing |
| title_sort | systematic evaluation of signal to noise ratio in variant detection from single cell genome multiple displacement amplification and exome sequencing |
| topic | Single cell sequencing Sparse cell sequencing Allele dropout Signal-to-noise ratio Whole exome sequencing NGS |
| url | http://link.springer.com/article/10.1186/s12864-018-5063-5 |
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