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Related Concept Videos

RNA-seq03:21

RNA-seq

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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
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Rare Event Detection Using Error-corrected DNA and RNA Sequencing
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DNA read count calibration for single-molecule, long-read sequencing.

Luis M M Soares1, Terrence Hanscom1, Donald E Selby1

  • 1Genomics and Computational Biology, Homology Medicines Inc, Bedford, MA, USA.

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|November 2, 2022
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Summary
This summary is machine-generated.

Pacific Biosciences and Oxford Nanopore Technologies sequencing methods were evaluated for quantifying DNA mixtures. PacBio showed length dependence, while Oxford Nanopore

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Area of Science:

  • Genomics
  • Molecular Biology
  • Bioinformatics

Background:

  • Accurate quantification of DNA mixtures with varying molecular lengths is crucial for applications like gene therapy vector analysis.
  • Short-read Next-Generation Sequencing (NGS) cannot sequence long gene therapy vectors individually.
  • Vector preparations may contain shorter DNA fragments that exhibit different sequencing behaviors.

Purpose of the Study:

  • To assess the suitability of Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) NGS platforms for quantitative analysis of DNA mixtures with diverse molecular lengths.
  • To compare different library preparation methods for their efficacy in quantitative DNA assessment.

Main Methods:

  • Two distinct library preparation methods were employed for both PacBio and ONT sequencing.
  • Equimolar length standards were created using Escherichia coli (E. coli) genomic DNA.
  • Quantitative analysis of read frequency and length dependence was performed.
  • Cubic spline smoothing was utilized for modeling read frequency.

Main Results:

  • Both PacBio library preparations demonstrated a consistent, albeit complex, dependence on DNA length.
  • The transposase-based ONT library preparation offered predictable length dependence but lost original length information due to random sequence starts.
  • The ligation-based ONT library preparation preserved length information but exhibited more variable read frequencies.
  • Differences in E. coli genomic copy number between exponential and stationary growth phases were detectable, indicating method sensitivity.
  • E. coli DNA served as a suitable internal spike-in for quantifying DNA fragments ranging from 200 bp to 10 kb.

Conclusions:

  • PacBio sequencing provides a viable approach for quantitative assessment of varying DNA lengths, with detectable sensitivity to copy number variations.
  • ONT ligation-based library preparation retains length information, making it suitable for quantitative analysis, while transposase-based methods are less ideal for length-dependent quantification.
  • The use of E. coli as an internal standard enables robust quality control for DNA preparations, including Adeno-Associated Virus (AAV) samples, across a significant size range.