Evaluation of pretreatment methods for high-throughput sequencing of fecal and tissue samples
10.3760/cma.j.cn112866-20211102-00190
- VernacularTitle:粪便和组织样本高通量测序前处理方法的评估
- Author:
Xiaohua MA
1
;
Sasa CHAI
;
Lili LI
;
Guisen ZHENG
;
Zhaojun DUAN
Author Information
1. 甘肃中医药大学公共卫生学院，兰州 730000
- Keywords:
high-throughput sequencing;
Virus;
Pretreatment;
Methodology;
Feces;
Tissue
- From:
Chinese Journal of Experimental and Clinical Virology
2022;36(1):103-110
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To optimize and evaluate the pretreatment method for high-throughput sequencing of fecal and tissue samples to improve the sensitivity of high-throughput sequencing in the study of virome.Methods:For fecal samples, five virus-positive samples that have been detoxified from feces were selected, mixed as the simulated samples, and filters made of different materials were used, different processing times were set for nucleases, and different kits were used to extract nucleic acid. For tissue samples, two virus-positive samples that have been detected in animal tissues, filter and nuclease treatment were used, and different extraction method were used to extract nucleic acid. TaqMan real-time PCR was used to quantitatively evaluate the impact of each treatment on the virus and the advantages and disadvantages were compared. We used the SYBR Green real-time PCR quantitative method to evaluate the removal effect of the above method on bacteria 16S rRNA and host 12S rRNA genome.Results:For fecal samples, the 0.22 μm PES filter showed a better filtering effect, and the PVDF material filter reduces the sample volume; 2 h nuclease digestion was better than 1 h digestion to remove bacteria, and the virus loss was less; the use of RPMK kits can effectively reduce bacteria, but the effect of extracting some viruses was poor, and the MVSK kit has a better effect of extracting viral nucleic acid. For tissue samples, 0.22 μm PES filter filtration, nuclease digestion for 1 h and VNAEK II kit extraction of nucleic acid were the best, Trizol LS combined with the RPMK method was better for gDNA removal, but the virus loss was larger. The virus loss of the whole process of the pretreatment method of feces and tissue samples was (1.7-3.0) Ct and (1.6-2.5) Ct, respectively.Conclusions:The optimal method for fecal samples was to filter with a PES filter, then digest with nuclease for 2 hours, and then extract nucleic acids using the MVSK kit; the optimal method for tissue samples was to filter with a PES filter, then perform 1 h nuclease digestion, and then use VNAEK II kit to extract nucleic acids.
