Cat-D: a targeted sequencing method for the simultaneous detection of small DNA mutations and large DNA deletions with flexible boundaries

We describe a novel assay method: reverse-transcription reverse-complement polymerase chain reaction (RT-RC-PCR), which rationalises reverse transcription and NGS library preparation into a single closed tube reaction. By simplifying the analytical process and cross-contamination risks, RT-RC-PCR presents disruptive scalability and economy while using NGS and LIMS infrastructure widely available across health service, institutional and commercial laboratories. We present a validation of RT-RC-PCR for the qualitative detection of SARS-CoV-2 RNA by NGS. The limit of detection is comparable to real-time RT-PCR, and no obvious difference in sensitivity was detected between extracted nasopharyngeal swab (NPS) RNA and native saliva samples. The end point measurement of RT-RC-PCR is NGS of amplified sequences within the SARS-CoV-2 genome; we demonstrated its capacity to detect different variants using amplicons containing delH69-V70 and N501Y, both of which emerged in the UK Variant of Concern B.1.1.7 in 2020. In summary, RT-RC-PCR has potential to facilitate accurate mass testing at disruptive scale and cost, with concurrent detection of variants of concern.

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Tumor DNA Mutations From Intraparenchymal Brain Metastases Are Detectable in CSF

JCO Precision Oncology ◽

10.1200/po.20.00292 ◽

2021 ◽

pp. 163-172

Author(s):

Stephanie Kim Cheok ◽

Azeet Narayan ◽

Anna Arnal-Estape ◽

Scott Gettinger ◽

Sarah B. Goldberg ◽

...

Keyword(s):

Brain Metastases ◽

Cohort Analysis ◽

Surrogate Marker ◽

Normal Pressure ◽

Tumor Evolution ◽

Leptomeningeal Disease ◽

Dna Mutations ◽

Sequencing Method ◽

Cancer Types ◽

Tumor Dna

PURPOSE Discordant responses between brain metastases and extracranial tumors can arise from branched tumor evolution, underscoring the importance of profiling mutations to optimize therapy. However, the morbidity of brain biopsies limits their use. We investigated whether cell-free DNA (cfDNA) in CSF could serve as an effective surrogate marker for genomic profiling of intraparenchymal (IP) brain metastases. METHODS CSF and blood were collected simultaneously from patients with progressive brain metastases undergoing a craniotomy or lumbar puncture. Mutations in both biofluids were measured using an error-suppressed deep sequencing method previously published by our group. Forty-three regions of 24 cancer-associated genes were assayed. RESULTS This study enrolled 14 patients with either IP brain metastases (n = 12) or cytology-positive leptomeningeal disease (LMD, n = 2) and two controls with normal pressure hydrocephalus. Primary cancer types were lung, melanoma, renal cell, and colorectal. cfDNA was measurable in all sixteen samples of CSF. Cancer-associated mutations were found in the CSF of ten patients (eight with IP [67%] and two with LMD [100%]) and plasma of five patients (five with IP [42%] and none with LMD). All patients with plasma cfDNA had extracranial tumors. Among the five patients in the cohort who also had mutation data from time-matched brain metastasis tissue, four patients (80%) had matching mutations detected in CSF and brain, whereas only one patient (20%) had matching mutations detected in plasma and brain. CONCLUSION The detection of mutational DNA in CSF is not restricted to LMD and was found in two thirds of patients with IP brain metastases in our cohort. Analysis of CSF can be a viable alternative to biopsy for detection of somatic mutations in brain metastases.

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Unique Associations of DNA Methylation Regions With 24-Hour Blood Pressure Phenotypes in Blacks

Hypertension ◽

10.1161/hypertensionaha.121.18584 ◽

2022 ◽

Author(s):

Michelle L. Roberts ◽

Theodore A. Kotchen ◽

Xiaoqing Pan ◽

Yingchuan Li ◽

Chun Yang ◽

...

Keyword(s):

Blood Pressure ◽

Dna Methylation ◽

Large Scale ◽

Cumulative Effect ◽

Targeted Sequencing ◽

Reduced Representation ◽

Gene Environment ◽

Reduced Representation Bisulfite Sequencing ◽

Sequencing Method ◽

Hypertension Development

Background: Epigenetic marks (eg, DNA methylation) may capture the effect of gene-environment interactions. DNA methylation is involved in blood pressure (BP) regulation and hypertension development; however, no studies have evaluated its relationship with 24-hour BP phenotypes (daytime, nighttime, and 24-hour average BPs). Methods: We examined the association of whole blood DNA methylation with 24-hour BP phenotypes and clinic BPs in a discovery cohort of 281 Blacks using reduced representation bisulfite sequencing. We developed a deep and region-specific methylation sequencing method, Bisulfite ULtrapLEx Targeted Sequencing and utilized it to validate our findings in a separate validation cohort (n=117). Results: Analysis of 38 215 DNA methylation regions (MRs), derived from 1 549 368 CpG sites across the genome, identified up to 72 regions that were significantly associated with 24-hour BP phenotypes. No MR was significantly associated with clinic BP. Two to 3 MRs were significantly associated with various 24-hour BP phenotypes after adjustment for age, sex, and body mass index. Together, these MRs explained up to 16.5% of the variance of 24-hour average BP, while age, sex, and BMI explained up to 11.0% of the variance. Analysis of one of the MRs in an independent cohort using Bisulfite ULtrapLEx Targeted Sequencing confirmed its association with 24-hour average BP phenotype. Conclusions: We identified several MRs that explain a substantial portion of variances in 24-hour BP phenotypes, which might be excellent markers of cumulative effect of factors influencing 24-hour BP levels. The Bisulfite ULtrapLEx Targeted Sequencing workflow has potential to be suitable for clinical testing and population screenings on a large scale.

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