Cell Reports：去除宿主和胞外DNA以提高微生物基因组得率（痰液样本）
The following article is from 三代测序 Author 很跩的土豆
简介
标题：Cell Reports：去除宿主和胞外DNA以提高微生物基因组得率（痰液样本）
杂志：Cell Reports
影响因子：8.109
发表时间：2019年2月19日
解读：很跩的土豆
编辑：很跩的土豆
导读：宏基因组测序是微生物鉴定和功能分析的重要手段。然而，宿主DNA的含量过高，微生物基因组得率较低是影响其分析的一大障碍；此外，呼吸道样本中的富集的微生物还能产生大量的来自生物膜或者死细胞的胞外DNA。本研究构建了一种通过真核细胞选择性裂解和内切酶酶切技术，以同时去除痰液样本中宿主和胞外DNA的方法。这种方法可增加微生物测序深度，同时可检测微生物数量、耐药基因等覆盖度也显著增加。
文章亮点：
（1）宿主和细菌胞外DNA可造成宏基因组测序的偏倚
（2）低张性溶解、核酸内切酶酶切能有效降低宿主和细菌胞外DNA
（3）降低胞外DNA可优化细菌活细胞DNA的宏基因组测序结果
（4）提高细菌测序结果的覆盖度可对某些重要基因进行检测
正文
一、方法
1. Mock community构建
从痰液样本中分离的七种细菌，Pseudomonas aeruginosa, Staphylococcus aureus, Neisseria sp. unclassified, Achromobacter xylosoxidans, Streptococcus salivarius, Stenotrophomonas maltophila 和 Rothia mucilaginosa，培养后Sanger测序。各种培养后的细菌以特定体积混合以形成mock community。相对丰度: 63.8% Pseudomonas, 29.5% Staphylococcus, 2.89% Neisseria, 0.398% Achromobacter, 1.02% Streptococcus, 2.04% Stenotrophomonas ，0.370% Rothia. 16S rRNA拷贝数矫正后，60.0% Pseudomonas, 34.3% Staphylococcus, 2.89% Neisseria, 0.398% Achromobacter, 1.02% Streptococcus, 2.04% Stenotrophomonas，0.370% Rothia。随后，即刻用于DNA提取。
胞外DNA的定量：离心培养基、收集上清，16S qPCR定量细菌总DNA。
2. DNA提取
（1）标准步骤：
1）200mg痰液样本或200ul mock community，1ml PBS重悬后13，000g离心3min；
2）400ul TE buffer重悬沉淀，1mm和0.1mm硅锆混合（silica:zirconia）beads、一颗碳化钨（tungsten-carbide）bead加入TE重悬液中，MiniBeadBeater破碎一分钟；随后，95℃ 5min。
3）加入Lysozyme (Sigma L6876, 3 mg/mL final) 和 lysostaphin (Ambi LSPN, 0.14 mg/mL final)，37℃孵育1h；
4）加入Proteinase K (Invitrogen 25530049, 1.4 mg/mL final) 和SDS (1.8% final) 56℃孵育30min，随后冷却至室温；
5)溶液转移至新馆，加入5M NaCl （2M final）；随后加入phenol:chloroform:isoamylalcohol (25:24:1)溶液，加入体积与原溶液体积1：1，随后RT 20min；
6）13，000g离心20min，收集上层清液，加入0.133倍体积浓度为7.5 M的乙酸铵（ammonium acetate），最溶液使用过100%乙醇1：1稀释，以获取DNA。
7）DNA转移至离心柱、清洗。
（2）去除抗体：1ug获取的DNA，使用NEBNext Microbiome DNA Enrichment Kit (NEB E2612S)进一步处理（见说明书）；
（3）细胞裂解：痰液使用1mlPBS重悬，13，000g离心20min，产物使用1ml TrypZean（Sigma T3449）和0.05% Tween-20（Sigma P9416）重悬，37℃孵育60min。溶液vortex后，5，000g离心2min弃上清，13，000g离心10min弃上清后PBS重悬，开始标准DNA提取步骤。
（4）Molysis method：采用Molzym’s Molysis Human DNA removal kit (D-300-050), 按照说明书处理痰液样本，在‘Buglysis’步骤暂停。随后使用PBS清洗、TE buffer 重悬，随后进行DNA标准提取流程。
（5）Benzonase 1 method：在痰液样本中加入7ml dH2O，室温轻柔震荡孵育1h。加入10x strength Benzonase buffer（200 mM Tris-HCl, 10 mM MgCl2）至终浓度为1X，再加入250U Benzonase (Sigma E-1014)，37℃轻柔震荡孵育2h。随后加入EDTA (5 mM final)和 NaCl (150 mM final)终止反应。终溶液8，000g离心10min，PBS清洗，400ulTE buffer重悬后进行DNA标准提取流程。
（6）Benzonase 2 method：与Benzonase 1不同的是，核酸酶孵育2h后，在加入EDTA终止反应前，8，000g离心10min，PBS清洗，400ulTE buffer重悬。随后加入EDTA (5 mM final)并直接进入标准DNA提取流程。
（7）PMA method：将300ulPBS、200ul mock community和1ul叠氮溴化丙锭（propidium monoazide，溶于水后浓度20mM）加入唾液样本中，室温孵育5min。随后在160LED灯下轻柔震荡孵育15min。13，000g离心10min，PBS清洗，400ulTE buffer重悬后进行DNA标准提取流程。
3. 其他（略）
二、结果
1. 宿主DNA的去除能力
Figure 1. Comparison of 4 Methods for Selective Depletion of Human DNA from CF Sputum Samples
DNA was extracted from 8 CF sputum samples with standard extraction and 4 different methods for selectively depleting human DNA.
(A) 宿主DNA占qPCR总DNA（含宿主和细菌DNA）的比例；
(B) Proportion of human to total reads, calculated by mapping all metagenomic sequencing reads to the human genome.
(C) Ratio of microbial metagenomic sequencing reads yielded by each extraction method compared to standard extraction.
(D) Total bacterial load yielded by each extraction method, calculated by qPCR targeting the 16S rRNA gene. The colored lines connect data points from the same sample, and the dotted line indicates the limit of detection.
Boxes represent the interquartile region, and black lines within boxes indicate the median value. Pair- wise Wilcoxon signed rank tests were used to compare results from selective human DNA depletion methods to standard extraction, with a Benjamini-Hochberg correction for multiple comparisons with all of the comparisons combined (A–D). No comparison reached the level of signifi- cance (p < 0.05).
2. 去除宿主DNA对检测到的菌群组成的影响
Figure 2. Effect of Extraction Method on Metagenomic Sequencing Taxonomic Profile and Viable Counts of the 8 Test Set 1 CF Sputum Samples
(A) Number of distinct genera detected by metagenomic sequencing from DNA prepared with each extraction method. Results from extractions, including selective depletion of human and extracellular DNA, were compared to standard extraction conditions. Boxes represent the interquartile region, and black lines indicate the median value. Pairwise Wilcoxon signed rank tests were performed comparing each extraction method to standard extraction with a Benjamini- Hochberg correction for multiple comparisons, with no comparison reaching the level of significance (p < 0.05).
(B) Difference in relative abundance identified by each extraction method (compared with standard extraction) for the 10 most abundant taxa. Each data point indicates an individual sample.
(C) Effect of benzonase treatment on viable P. aeruginosa counts. Cultures of 6 separate P. aeruginosa clinical isolates were subjected to both benzonase1 and benzonase2 processing methods, which differ only in the order in which EDTA is added. Viable counts were measured after the hypotonic, host-cell lysis step and after enzyme inactivation, as indicated.
3. Benzonase2 方法对细菌宏基因组和16S rRNA测序结果的影响
Figure 3. Sequence-Based Phylogenetic Composition of DTT-Treated Test Set 2 Samples after Standard and Benzonase Extraction
DNA extracted from each of 4 DTT-treated, homogenized CF sputum samples was analyzed using both metagenomic sequencing (MetaPhlAn2, top row) and 16S amplicon sequencing (16S Amplicon, bottom row).
Figure 4. Effect of the Refined Benzonase2 Extraction Method on Selective Human DNA Depletion and Microbial Sequencing Depth
Total DNA from the 4 test set 2 sputum samples was extracted using standard, benzonase1, and benzonase2 extraction methods and analyzed to show the following.
(A) Proportion of human DNA relative to total DNA as determined by qPCR.
(B) Proportion of human to total reads calculated by mapping all of the metagenomic sequencing reads to a reference human genome.
(C) Ratio of microbial shotgun sequencing reads yielded by each extraction method compared to the standard extraction.
(D) Total bacterial load (genome equivalents [GEs]) yielded from each extraction method, as deter- mined by qPCR targeting the 16S rRNA gene. Boxes represent the interquartile region, and black lines indicate the median value.
(E and F) Number of genera detected in each extract using (E) metagenomic sequencing or (F) 16S amplicon sequencing. Each color represents a different sample.
Results from each extraction method were compared to the standard extraction conditions using pairwise, 2-sided t tests with a Benjamini- Hochberg correction for multiple comparisons, identifying no significant differences.
4. 基于核酸酶提取细菌基因组可很好的反映菌群组成的准确性、多样性
Figure 5. Effect of Extraction Method on Sequencing-Based Taxonomic Profile of a Bacterial Mock Community
Data represent a single mock community extracted in parallel with each extraction method. Phylogenetic tree constructed using Bray-Curtis dissimilarity (‘‘Tree’’) and the corresponding phylogenetic composition (‘‘Community Composition’’) determined via (left) metagenomic sequencing and (right) 16S amplicon sequencing. Input refers to the relative abundance of the mock community before extraction based on quantitative culture. Viable counts were corrected for 16S copy number in the analysis of 16S amplicon sequence data.
5. 选择性去除宿主DNA可增加宏基因组测序过程中菌群基因的覆盖度
Figure 6. Increase in Microbial Sequence Coverage after Human DNA Depletion of the 4 Test Set 2 Sputum Samples
(A) Contigs were assembled using the same 4 sputum samples and processing methods as in Figures 3 and 4, and all of the reads were subsequently mapped back onto these contigs. Each radial line represents a single contig ordered by Euclidean distance based on sequence content. The height of each bar of darker shading represents the average coverage across that contig (average of the sequence coverage of each nucleotide across a given contig) in a given sample from 0 to 103. Radial black lines in the outermost ring indicate contigs annotated as human.
(B) Average coverage of 145 identified antibiotic resistance genes in each sample as a proportion of 103 coverage. Each radial line represents an individual antibiotic resistance gene. Three genes described in the text are indicated. The height of the bars represents mean coverage for each gene, from 0 to 103.
三、总结
Benzonase2方法是一个改进的微生物基因组的提取方法，能够提高宏基因组的测序质量并保持微生物组成的准确性和覆盖度。
索引
往期文献：【文献解读】新冠病毒病毒活性与COVID-19患者肠道菌群的关系
【文献解读】Protein Cell：扩增子和宏基因组数据分析实用指南
【文献解读】SciRep：ONT MinION和Illumina Miseq对室内尘埃微生物组16S rRNA测序的区别
参考
[1] Nelson et al., 2019, Cell Reports 26, 2227–2240
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