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Sequencing of mRNA from Whole Blood using Nanopore Sequencing
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CarrierSeq: a sequence analysis workflow for low-input nanopore sequencing.

Angel Mojarro1, Julie Hachey2, Gary Ruvkun3

  • 1Department of Earth, Atmospheric and Planetary Sciences, Massachusetts Institute of Technology, 77 Massachusetts Ave, E25-610, Cambridge, MA, 02139, USA. mojarro@mit.edu.

BMC Bioinformatics
|March 29, 2018
PubMed
Summary
This summary is machine-generated.

Carrier sequencing enables low-input DNA analysis using nanopore sequencing by adding a genomic carrier. This method, with the CarrierSeq workflow, successfully identifies target DNA from low-biomass samples, overcoming previous limitations.

Keywords:
Low-input sequencingMetagenomicsNanopore sequencing

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Area of Science:

  • Genomics
  • Bioinformatics
  • Molecular Biology

Background:

  • Long-read nanopore sequencing is crucial for species-level taxonomic identification.
  • Low-biomass environmental samples often yield insufficient DNA for standard nanopore sequencing input requirements.
  • This limitation hinders
  • sample to sequence
  • metagenomics for challenging samples.

Purpose of the Study:

  • To develop a method for sequencing low-input DNA using nanopore technology.
  • To enable metagenomic analysis from low-biomass or recalcitrant environmental samples.
  • To address the DNA input limitations of current nanopore sequencing protocols.

Main Methods:

  • Carrier sequencing: preparing low-input DNA with a genomic carrier for optimal library preparation and sequencing stoichiometry without amplification.
  • CarrierSeq workflow: a bioinformatics pipeline to identify target DNA reads from the genomic carrier.
  • Experimental validation using a mixture of Bacillus subtilis DNA and Enterobacteria phage λ DNA.

Main Results:

  • Successfully sequenced and identified low-input Bacillus subtilis DNA (0.2 ng) in a high background of carrier DNA (1000 ng).
  • Detected target reads, contamination reads, and "high quality noise reads" (HQNRs) after filtering.
  • Identified HQNRs as potential nanopore sequencing artifacts associated with specific channels.

Conclusions:

  • Carrier sequencing and the CarrierSeq workflow effectively enable nanopore sequencing of low-input DNA.
  • A statistical method, treating sequencing as a Poisson arrival process, can reject data from channels with high HQNRs.
  • This approach allows for the retention of low-input target reads, improving data quality and usability.