Use of synthetic DNA spike-in controls (sequins) for human genome sequencing

Blackburn, James; Wong, Ted; Madala, Bindu Swapna; Barker, Chris; Hardwick, Simon A.; Reis, André; Mercer, Tim R.

doi:10.1038/s41596-019-0175-1

Cited by 24 publications

(38 citation statements)

References 37 publications

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“…To overcome the shortage of quality control materials [ 6 – 8 ], we developed a set of suitable quality-control cells that comprise the common aneuploidies trisomy 13, 21, and 18, and 47,XXY based on immortalization of amniocytes by SV40LT-transfection [ 15 ] (Fig. 1 A-B).…”

Section: Discussionmentioning

confidence: 99%

“…In general, the most important properties for quality control materials are as follows: they must behave like the real samples, and they must be available in sufficient quantity for developing internal quality control (IQC) charts to be used for monitoring quality in real time. At present, standardized IQCs are not easily available for prenatal molecular diagnosis because Levey-Jennings charts have not been used in molecular biology laboratories, and there is an insufficient supply of characterized paired materials such as cells with trisomy 18 and 13 in the cell line repository [ 6 – 8 ].…”

Section: Introductionmentioning

confidence: 99%

“…In the current study, we developed a set of aneuploidy chimeric quality-control cells (A-H) by immortalizing aneuploid amniocytes via SV40LTtransfection to address the lack of quality-control materials for methods used in prenatal diagnosis such as NIPT, NGS, and FISH [ 6 – 8 ]. More importantly, we developed a Levey-Jennings chart-based IQC system for detecting fatal aneuploidies by FISH by testing these quality-control cells(A-H) and clinical samples under the same experimental conditions.…”

Section: Introductionmentioning

confidence: 99%

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A novel use for Levey-Jennings charts in prenatal molecular diagnosis

Weng

Ying

et al. 2020

BMC Med Genomics

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Background: The goal of this study was to determine whether Levey-Jennings charts, which are widely used in clinical laboratories, can be used to create standardized internal quality controls (IQCs) for prenatal molecular diagnosis. Methods: Aneuploid amniocyte lines with trisomy 13, 21, and 18, and 47,XXY were established by transfection with SV40LTag-pcDNA3.1(−)and combined at different ratios to generate aneuploidy chimeric quality-control cell mixtures A to H. These quality-control cells were then used to calculate the X, X ±1 standard deviation (SD), X ±2 SD, and X ±3 SD values to develop standardized IQCs for methods used for the prenatal diagnosis of aneuploidies such as FISH. Results: Methods for constructing aneuploid amniocyte lines were developed and a set of quality-control cells (A-H) were prepared. The X ±1 SD, X ±2 SD, and X ±3 SD values of these quality-control cells for trisomy 13 and 21

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A novel use for Levey-Jennings charts in prenatal molecular diagnosis

Weng

Ying

et al. 2020

BMC Med Genomics

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“…The addition of spike-in controls dramatically changes the interpretation of RNA-seq, chromatin immunoprecipitationsequencing (ChIP-seq), and other genomic assay results. [3][4][5][6][7] As such, all quantitative genome-wide assays would benefit from the addition of spike-in controls. 4 The most common approach to normalizing sequencing assay data consists in dividing the number of reads at each genomic region by the total number of reads genome-wide.…”

Section: Introductionmentioning

confidence: 99%

Sensitive and reproducible cell-free methylome quantification with synthetic spike-in controls

Wilson

Shen

Harmon

et al. 2021

Preprint

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BACKGROUND: Cell-free methylation DNA immunoprecipitation-sequencing (cfMeDIP-seq) identifies genomic regions with DNA methylation, using a protocol adapted to work with low-input DNA samples and with cfDNA. This method allows for DNA methylation profiling of circulating tumour DNA in cancer patients' blood samples. Such epigenetic profiling of circulating tumour DNA provides information about in which tissues tumour DNA originates, a key requirement of any test for early cancer detection. In addition, DNA methylation signatures provide prognostic information and can detect relapse. For robust quantitative comparisons between samples, immunoprecipitation enrichment methods like cfMeDIP-seq require normalization against common reference controls. METHODS: To provide a simple and inexpensive reference for quantitative normalization, we developed a set of synthetic spike-in DNA controls for cfMeDIP-seq. These controls account for technical variation in enrichment efficiency due to biophysical properties of DNA fragments. Specifically, we designed 54 DNA fragments with combinations of methylation status (methylated and unmethylated), fragment length (80 bp, 160 bp, 320 bp), G+C content (35%, 50%, 65%), and fraction of CpG dinucleotides within the fragment (1/80 bp, 1/40 bp, 1/20 bp). We ensured that the spike-in synthetic DNA sequences do not align to the human genome. We integrated unique molecular indices (UMIs) into cfMeDIP-seq to control for differential amplification after enrichment. To assess enrichment bias according to distinct biophysical properties, we conducted cfMeDIP-seq solely on spike-in DNA fragments. To optimize the amount of spike-in DNA required, we added varying quantities of spike-in control DNA to sheared HCT116 colon cancer genomic DNA prior to cfMeDIP-seq. To assess batch effects, three separate labs conducted cfMeDIP-seq on peripheral blood plasma samples from AML patients. RESULTS: We show that cfMeDIP-seq enriches for highly methylated regions, capturing ≥99.99% of methylated spike-in control fragments with ≤0.01% non-specific binding and preference for both high G+C content fragments and fragments with more CpGs. The use of 0.01 ng of spike-in control DNA total provided sufficient sequencing reads to adjust for variance due to fragment length, G+C content, and CpG fraction. Using the known amount of each spiked-in fragment, we created a generalized linear model that absolutely quantifies molar amount from read counts across the genome, while adjusting for fragment length, G+C content, and CpG fraction. Employing our spike-in controls greatly mitigates batch effects, reducing batch-associated variance to ≤1% of the total variance within the data. DISCUSSION: Incorporation of spike-in controls enables absolute quantification of methylated cfDNA generated from MeDIP-seq experiments. It mitigates batch effects and corrects for biases in enrichment due to known biophysical properties of DNA fragments and other technical biases. We created an R package, spiky, to convert read counts to picomoles of DNA fragments, while adjusting for fragment properties that affect enrichment. The spiky package is available on GitHub (https://github.com/trichelab/spiky) and will soon be available on Bioconductor.

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“…Synthetic DNAs of known sequence can be used as a standard for validation of a DNA sequencer itself [14], but it is desirable to use a pathological specimen closer to real clinical samples in order to validate the whole processes of diagnosis including sample preparation [11]. When the number of gene to be sequenced is limited, a portion of the clinical specimen can be preserved and used as the reference material for the specific gene, but it is difficult to prepare such standards for all the genes in the panel.…”

Section: Introductionmentioning

confidence: 99%

Preparation of the standard cell lines for reference mutations in cancer gene-panels by genome editing in HEK 293 T/17 cells

et al. 2020

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Background: Next Generation Sequencer (NGS) is a powerful tool for a high-throughput sequencing of human genome. It is important to ensure reliability and sensitivity of the sequence data for a clinical use of the NGS. Various cancer-related gene panels such as Oncomine™ or NCC OncoPanel have been developed and used for clinical studies. Because these panels contain multiple genes, it is difficult to ensure the performance of mutation detection for every gene. In addition, various platforms of NGS are developed and their cross-platform validation has become necessity. In order to create mutant standards in a defined background, we have used CRISPR/Cas9 genome-editing system in HEK 293 T/17 cells. Results: Cancer-related genes that are frequently used in NGS-based cancer panels were selected as the target genes. Target mutations were selected based on their frequency reported in database, and clinical significance and on the applicability of CRISPR/Cas9 by considering distance from PAM site, and off-targets. We have successfully generated 88 hetero-and homozygous mutant cell lines at the targeted sites of 36 genes representing a total of 125 mutations. Conclusions: These knock-in HEK293T/17 cells can be used as the reference mutant standards with a steady and continuous supply for NGS-based cancer panel tests from the JCRB cell bank. In addition, these cell lines can provide a tool for the functional analysis of targeted mutations in cancer-related genes in the isogenic background.

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Use of synthetic DNA spike-in controls (sequins) for human genome sequencing

Cited by 24 publications

References 37 publications

A novel use for Levey-Jennings charts in prenatal molecular diagnosis

A novel use for Levey-Jennings charts in prenatal molecular diagnosis

Sensitive and reproducible cell-free methylome quantification with synthetic spike-in controls

Preparation of the standard cell lines for reference mutations in cancer gene-panels by genome editing in HEK 293 T/17 cells

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