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    Home > Biochemistry News > Biotechnology News > Nat Biotech: Same-day identification of pathogenic variants with ultrafast nanopore sequencing in just 8 hours

    Nat Biotech: Same-day identification of pathogenic variants with ultrafast nanopore sequencing in just 8 hours

    • Last Update: 2022-04-21
    • Source: Internet
    • Author: User
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    A team of researchers led by Stanford University recently demonstrated the value of rapid whole-genome sequencing in the diagnosis of critical illness
    .
    They have developed an ultra-fast nanopore sequencing process that can yield clues to genetic diseases in eight hours, which used to take days

    .

    The researchers developed the process based on Oxford Nanopore's high-throughput sequencing device PromethION 48, combined with an optimized sample preparation scheme, parallel sequencing on 48 sequencing chips, and rapid data analysis, which is expected to help doctors quickly diagnose critically ill patients.

    .
    The results were published in the journal Nature Biotechnology on March 28

    .

    This time, 12 critically ill patients, ranging in age from 3 months to 57 years old, participated in the study.
    They were all critically ill, but doctors were at a loss to determine the cause

    .
    A team led by Euan A.
    Ashley, Distinguished Professor at Stanford University School of Medicine, has partnered with companies such as Oxford Nanopore, Nvidia, and Google to shorten the time it takes to sequence the entire human genome

    .

    The ultra-fast sequencing and analysis pipeline was completed in one day for all participants, and the causative genetic variant was identified in five patients
    .
    This indicates a diagnosis rate of 42%

    .
    The researchers believe that the rapid identification of pathogenic variants can provide guidance for clinical management, and it is also expected to improve prognosis and reduce the cost of treatment

    .

    Ultra-fast sequencing workflow

    Whole-genome sequencing has advantages in medical diagnosis, but the pipeline for sequencing and downstream analysis is often slow
    .
    In 2015, the fastest time needed for sequencing and diagnosis was 26 hours

    .
    In the past two years, this time has been further shortened to more than ten hours

    .
    Of course, researchers also face a series of problems, such as the inability of traditional sample preparation protocols to generate libraries from limited blood, which is especially important when analyzing critically ill neonates

    .
    In addition, the speed of data generation far exceeds the speed of base calling and alignment

    .

    To this end, Ashley-led research teams have developed a genome-wide nanopore sequencing pipeline that improves library preparation, uses a cloud-based module for near real-time base calling and alignment, and enables rapid variant calling (including SNPs) , indel and SV) and focused variant filtering
    .

    The researchers used Oxford Nanopore's high-throughput sequencing device PromethION 48 to carry out whole-genome sequencing
    .
    In their validation work, they sequenced the NIST reference material HG002 genome

    .
    They sequenced a single sample in parallel on 48 sequencing chips and obtained the complete human genome sequence in just two hours

    .

    They found that calling performance for small variants was similar regardless of whether barcodes were used, and similar results were observed for structural variants
    .
    Based on these data, the team chose not to use barcodes, which reduced library preparation time by 37 minutes and improved downstream sequencing efficiency

    .

    At the same time, sequencing speed did not affect the accuracy of variant calling
    .
    The results showed that when HG002 was used for the benchmark, the F1 score of SNP was 0.
    9974 and the F1 score of indel was 0.
    7322

    .
    On average, they found 4.
    49 million small fragment variants and 22 structural variants that were prioritized for analysis in each sample

    .

    The research team tackled the data analysis problem by developing a cloud computing architecture (Google Cloud Platform) and parallel base calling and alignment across multiple GPUs (NVIDIA GPUs)
    .
    Using these methods to analyze deep genome coverage (200 Gb), they obtained long-read whole human genomes in near real-time

    .

    Application in clinical setting

    To illustrate how the procedure performs in a real-world setting, the researchers specifically discussed two patients
    .
    The first case was a 57-year-old man with a number of health problems, including hyperthyroidism, high blood pressure, severe COVID-19, and the need for bilateral lung transplants

    .
    Cardiac magnetic resonance images provide evidence of hypertrophic cardiomyopathy, but the differential diagnosis is broad and requires rapid molecular testing to confirm the diagnosis

    .

    The researchers completed variant calling in less than 7 hours and found 4.
    31 million small segment variants and 35,800 structural variants

    .
    They then identified a diagnostic heterozygous variant in the TNNT2 gene and classified it as a likely pathogenic variant according to ACMG guidelines, reducing the need for multiple subsequent testing and cardiac biopsies

    .
    The whole process took 7 hours and 18 minutes

    .

    The second patient, a 14-month-old baby girl with dystonic posture and developmental delay, was admitted to the Pediatric Intensive Care Unit after experiencing cardiac arrest and respiratory failure
    .
    Seven hours after the blood was drawn, they obtained 4.
    48 million small fragment variants and 36,400 structural variants

    .
    After filtering, 31 small variants and 21 structural variants were given priority to manual review

    .
    They then found a candidate variant of unknown significance in the LZTR1 gene

    .

    For this case, the time to acquire 200 Gb of sequencing data was 2 hours and 46 minutes
    .
    Gordon Sanghera, CEO of Oxford Nanopore, commented: "2.
    5 hours, 60x coverage is equivalent to 2.
    5 minutes for human whole genome sequencing

    .
    We believe we can simplify this process and cut the time in half

    .
    This work illustrates the rapid genome Sequencing has the potential to fundamentally change the future of critical care

    .
    "

    "We are delighted to see the publication of this pivotal work and demonstrate the potential impact of ultrafast nanopore sequencing in critically ill patients
    .
    Compared to standard diagnostic methods that can take weeks, this study provides clinicians with Accurate information so they can make decisions and be able to provide patients and their families with answers that day," he said

    .

    "Using whole genome sequencing to rapidly diagnose critically ill patients is challenging, but we have a solution," study participant Dr.
    John Gorzynski of Stanford University wrote on social media.

    The cost of this ultra-fast method, which includes DNA extraction, library preparation, sequencing, and data analysis, is estimated to be just over $5,000, including $1,600 for the flow chip, $1,584 for the nanopore sequencing reagents, and $568 for the analysis
    .
    Oxford Nanopore expects these costs to drop significantly as sample preparation, computing and sequencing chips improve

    .

    Original text retrieval

    Goenka, SD, Gorzynski, JE, Shafin, K.
    et al.
    Accelerated identification of disease-causing variants with ultra-rapid nanopore genome sequencing.
    Nat Biotechnol (2022).
    https://doi.
    org/10.
    1038/s41587-022- 01221-5

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