High Sensitivity of Shotgun Metagenomic Sequencing in Colon Tissue Biopsy by Host DNA Depletion

Medical Laboratory Science Gastroenterology Medicine Genetics Life Sciences Microbiology
host DNA depletion colon tissue biopsy shotgun metagenomic sequencing microbiota species discovery disease progression

High sensitivity of high-throughput metagenomic sequencing in colon tissue biopsies: Eliminating the impact of host DNA

Background

Evaluating bacterial taxonomic structure through next-generation sequencing without culturing has become a common method for studying the relationship between bacterial imbalance and various diseases. Previous studies have analyzed microbial community profiles of human oral, intestinal mucosa, and fecal samples through 16S rRNA gene amplicon or metagenomic sequencing. However, 16S rRNA gene sequencing has limitations in taxonomic identification resolution, while metagenomic sequencing can identify bacteria to species or even subspecies levels. Moreover, metagenomic sequencing can provide multi-kingdom data and infer interactions across multiple domains.

Although metagenomic sequencing has important clinical applications in characterizing microbial communities in tissue samples, including disease diagnosis and treatment, the majority of DNA in samples is human, limiting the detection of low-abundance bacteria. This imbalance in DNA abundance reduces the sensitivity of metagenomic sequencing in tissue samples. Therefore, to address this issue, researchers proposed an optimized method to improve the effectiveness of metagenomic sequencing in colon biopsy samples by removing host DNA.

Paper Source

This study was conducted by a research team from the State Key Laboratory of Digestive Disease at the Chinese University of Hong Kong, with main authors including Wing Yin Cheng, Wei-Xin Liu, Yanqiang Ding, Guoping Wang, Yu Shi, and others. The article was published in Volume 21 (2023) of “Genomics, Proteomics & Bioinformatics”.

Research Process

In this study, researchers conducted group experiments on human or mouse colon biopsy samples, with one group undergoing host DNA removal and the other serving as a control. Host DNA was removed through differential lysis of mammalian and bacterial cells, followed by metagenomic sequencing of the samples. The research process consisted of the following steps:

  1. Sample homogenization and grouping: Human and mouse colon tissues were homogenized and divided equally into two parts, one for host DNA removal and the other as a control group.

  2. Differential lysis of host cells:

    • For the host removal group, mammalian cells were first lysed to release host DNA, which was then degraded by Benzonase enzyme.
    • Subsequently, bacterial cells were lysed to extract bacterial DNA for metagenomic sequencing.

  3. Metagenomic sequencing: Paired-end 150 bp (PE150) sequencing was performed using the Illumina HiSeq 2000 platform. Sequencing data was aligned with reference genome databases for taxonomic analysis.

  4. Data analysis:

    • Trimmomatic was used to filter sequencing read quality.
    • End-to-end alignment was performed using Bowtie-2 to remove host sequences.
    • Remaining non-host reads were aligned to bacterial genome databases using the Kraken classification software.

Research Results

The study showed that host DNA removal significantly increased bacterial reads and the number of species discovered. In human and mouse colon tissue samples, after host DNA removal, bacterial sequence reads increased by 2.46 ± 0.20 fold and 5.46 ± 0.42 fold, respectively, while host reads decreased by 6.80% ± 1.06% and 10.2% ± 0.83%. Furthermore, the number of detected bacterial species significantly increased after host DNA removal, with 2998 ± 401 species detected in human samples compared to 891 ± 98 in the control group; in mouse samples, 3707 ± 1465 species were detected compared to 1555 ± 314 in the control group. Most bacterial species from the non-removal group (93.45% ± 0.89% for humans, 83.34% ± 7.00% for mice) were also detected in the removal group, indicating that the method improved species detection sensitivity without compromising microbial composition. Overall, host DNA removal significantly increased bacterial detection coverage and diversity in tissue samples.

Additionally, host DNA removal increased the coverage of bacterial genes. Gene accumulation analysis showed a 33.89% increase in bacterial gene detection in human colon biopsy samples and a 95.75% increase in mouse samples.

Conclusion and Value

The results demonstrate that the optimized host DNA removal method can improve the sensitivity of metagenomic sequencing and bacterial detection coverage while maintaining the relative diversity of microbial communities. This method performs excellently on tissue biopsy samples and has important clinical application value. For example, it can identify bacteria important for disease onset or progression, providing a basis for early diagnosis and treatment. In cases where limited microsamples are available, this method can preserve more samples for other analyses such as transcriptomics, proteomics, and metabolomics. Furthermore, the method is easily applicable to clinical sample processing, especially in studying the relationship between gut microbiota and host health.

Research Highlights

  1. Host DNA removal technology significantly improved the sensitivity and species detection range of metagenomic sequencing.
  2. The relative composition of microbial communities was maintained without significant structural changes.
  3. The extracted bacterial genes showed significant improvements in quantity and diversity.
  4. The method has a wide range of applications and is significant for clinical sample processing.

Further Research

Future studies can continue to optimize the host DNA removal method for different types of tissue samples and explore its potential and limitations in other clinical applications. Researchers can also investigate the effectiveness of this method in different disease states to further advance research and clinical applications in this field.