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LABS: linear amplification-based bisulfite sequencing for ultrasensitive cancer detection from cell-free DNA

ABSTRACT: LABS: linear amplification-based bisulfite sequencing for ultrasensitive cancer detection from cell-free DNA

PROVIDER: PRJNA771691 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR16356326.fastq.gz	Fastqsanger.gz
	SRR16356327.fastq.gz	Fastqsanger.gz
	SRR16356328.fastq.gz	Fastqsanger.gz
	SRR16356329.fastq.gz	Fastqsanger.gz
	SRR16356330.fastq.gz	Fastqsanger.gz

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LABS: linear amplification-based bisulfite sequencing for ultrasensitive cancer detection from cell-free DNA

Project description:Methylation-based liquid biopsies show promise in detecting cancer from circulating cell-free DNA, but current limitations impede clinical application. Most assays necessitate substantial DNA inputs, posing challenges. Underrepresented tumor DNA fragments may go undetected during exponential amplification steps of traditional sequencing methods. Here we report LABS (Linear Amplification based Bisulfite Sequencing), enabling linear amplification of bisulfite-treated DNA fragments in a genome-wide, unbiased fashion, detecting cancer abnormalities with sub-nanogram inputs. Applying LABS to 100 patient samples revealed cancer-specific patterns, copy number alterations, and enhanced cancer detection accuracy by identifying tissue-of-origin and immune cell composition.

2024-04-24 | GSE186007 | GEO

Validation of the linear amplification of single bovine embryos

Project description:To generate expression profiles from single embryos, a protocol for linear amplification of RNA was developed on the basis of Baugh et al. (15). Linear amplification enabled production of sufficient amounts of aRNA for the microarray experiments and has been shown to reliably amplify the initial mRNA population (15, 18-20). Approximately 5 ng of total RNA can be isolated from a blastocyst-stage embryo (21); therefore, it was necessary to carry out multiple rounds of amplification. To test the reproducibility and fidelity of the amplification procedure, a validation experiment was conducted using RNA from the kidney tissue of a newborn calf. Two replicates of the bovine kidney RNA were amplified and hybridized to the microarray. Comparisons were made between aRNA (rounds 1, 2, and 3) and unamplified RNA. Keywords: cDNA microarray

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Effect of ligase on T7 based RNA linear amplification

Project description:Zhao et al. Amplification Table 3 This experiment was designed to evaluate the effect of ligase used in the second strand cDNA synthesis on the fidelity of T7 based RNA linear amplification. BC2 total RNA was amplified with or without ligase using Affymetrix protocol. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set

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Effect of column cleanup on T7 based RNA linear amplification

Project description:Zhao et al. Amplification Table 4 This experiment was designed to evaluate the effect of column cleanup on the fidelity and yield of T7 based RNA linear amplification. BC2 total RNA was amplified using the Jeffrey lab protocol with or without G50 cleanup. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set

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Single-tube Linear DNA amplification (LinDA) for robust ChIP-seq

Project description:Genome-wide profiling of transcription factors based on massive parallel sequencing of immunoprecipitated chromatin (ChIP-seq) requires nanogram amounts of DNA. Here we describe a high-fidelity, single-tube linear DNA amplification method (LinDA) for ChIP-seq and reChIP-seq with picogram DNA amounts obtained from a few thousand cells. This amplification technology will facilitate global analyses of transcription-factor binding and chromatin with very small cell populations, such as stem or cancer-initiating cells.

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Single-tube Linear DNA amplification (LinDA) for robust ChIP-seq

Project description:Single-tube Linear DNA amplification (LinDA) for robust ChIP-seq

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Maximizing linear amplification of nucleic acids with optimized T7 promoters

Project description:Maximizing linear amplification of nucleic acids with optimized T7 promoters

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Comparison of linear and exponential amplification techniques for expression profiling at the single-cell level

Project description:We tested the performance of three methods for amplifying single-cell amounts of RNA under ideal conditions: T7-based in vitro transcription; switching mechanism at 5' end of RNA template (SMART) PCR amplification; and global PCR amplification. All methods introduced amplification-dependent noise when mRNA was amplified 108-fold, compared with data from unamplified cDNA. PCR-amplified cDNA demonstrated the smallest number of differences between two parallel replicate samples and the best correlation between independent amplifications from the same cell type, with SMART outperforming global PCR amplification. SMART had the highest true-positive rate and the lowest false-positive rate when comparing expression between two different cell types, but had the lowest absolute discovery rate of all three methods. Direct comparison of the performance of SMART and global PCR amplification on single-cell amounts of total RNA and on single neural stem cells confirmed these findings. Under the conditions tested, PCR amplification was more reliable than linear amplification for detecting true expression differences between samples. SMART amplification had a higher true-positive rate than global amplification, but at the expense of a considerably lower absolute discovery rate and a systematic compression of observed expression ratios. Keywords: Oliginucleotide expression microarrays, T7-based linear amplification; SMART PCR-based amplification; global PCR amplification

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Effect of ligase on T7 based RNA linear amplification

2005-11-03 | E-GEOD-3561 | biostudies-arrayexpress

Optimization and evaluation of T7 based RNA linear amplification protocols for cDNA microarray analysis

Project description:Abstract: BACKGROUND: T7 based linear amplification of RNA is used to obtain sufficient antisense RNA for microarray expression profiling. We optimized and systematically evaluated the fidelity and reproducibility of different amplification protocols using total RNA obtained from primary human breast carcinomas and high-density cDNA microarrays. RESULTS: Using an optimized protocol, the average correlation coefficient of gene expression of 11,123 cDNA clones between amplified and unamplified samples is 0.82 (0.85 when a virtual array was created using repeatedly amplified samples to minimize experimental variation). Less than 4% of genes show changes in expression level by 2-fold or greater after amplification compared to unamplified samples. Most changes due to amplification are not systematic both within one tumor sample and between different tumors. Amplification appears to dampen the variation of gene expression for some genes when compared to unamplified poly(A)+ RNA. The reproducibility between repeatedly amplified samples is 0.97 when performed on the same day, but drops to 0.90 when performed weeks apart. The fidelity and reproducibility of amplification is not affected by decreasing the amount of input total RNA in the 0.3-3 micrograms range. Adding template-switching primer, DNA ligase, or column purification of double-stranded cDNA does not improve the fidelity of amplification. The correlation coefficient between amplified and unamplified samples is higher when total RNA is used as template for both experimental and reference RNA amplification. CONCLUSION: T7 based linear amplification reproducibly generates amplified RNA that closely approximates original sample for gene expression profiling using cDNA microarrays. This SuperSeries is composed of the SubSeries listed below.

2006-03-07 | GSE4349 | GEO

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