Amplicon sequencing with internal standards yields accurate picocyanobacteria cell abundances as validated with flow cytometry.

IF 5.1 Q1 ECOLOGY
ISME communications Pub Date : 2024-09-25 eCollection Date: 2024-01-01 DOI:10.1093/ismeco/ycae115
Alexandra E Jones-Kellett, Jesse C McNichol, Yubin Raut, Kelsy R Cain, François Ribalet, E Virginia Armbrust, Michael J Follows, Jed A Fuhrman
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Abstract

To understand ecosystem state and function, marine microbial ecologists seek measurements of organismal abundance and diversity at high taxonomic resolution. Conventional flow cytometry accurately estimates microbial cell abundance but only discerns broad groups with distinct optical properties. While amplicon sequencing resolves more comprehensive diversity within microbiomes, it typically only provides relative organismal abundances within samples, not absolute abundance changes. Internal genomic standards offer a solution for absolute amplicon-based measures. Here, we spiked genomic standards into plankton samples from surface seawater, gathered at 46-km intervals along a cruise transect spanning the southern California Current System and the oligotrophic North Pacific Subtropical Gyre. This enabled evaluation of the absolute volumetric gene copy abundances of 16S rRNA amplicon sequence variants (amplified with 515Y-926R universal primers, quantitatively validated with mock communities) and cell abundances of picocyanobacteria with known genomic 16S copy numbers. Comparison of amplicon-derived cell abundances of Prochlorococcus and Synechococcus with flow cytometry data from nearby locations yielded nearly identical results (slope = 1.01; Pearson's r = 0.9942). Our findings show that this amplicon sequencing protocol combined with genomic internal standards accurately measures absolute cell counts of marine picocyanobacteria in complex field samples. By extension, we expect this approach to reasonably estimate volumetric gene copies for other amplified taxa in these samples.

经流式细胞仪验证,使用内部标准进行扩增子测序可获得准确的皮蓝藻细胞丰度。
为了了解生态系统的状态和功能,海洋微生物生态学家寻求以高分类分辨率测量生物体的丰度和多样性。传统的流式细胞仪能准确估计微生物细胞的丰度,但只能分辨出具有不同光学特性的大类。虽然扩增子测序能解析微生物组内更全面的多样性,但它通常只能提供样本内的相对生物丰度,而不能提供绝对丰度变化。内部基因组标准为基于扩增片段的绝对测量提供了一种解决方案。在这里,我们在表层海水浮游生物样本中添加了基因组标准,这些样本是沿着横跨南加州洋流系统和低营养型北太平洋亚热带环流的巡航横断面以 46 公里的间隔采集的。这样就可以评估 16S rRNA 扩增子序列变体(使用 515Y-926R 通用引物扩增,并通过模拟群落进行定量验证)的绝对体积基因拷贝丰度和已知基因组 16S 拷贝数的皮蓝藻细胞丰度。将扩增片段推导出的原绿球藻和 Synechococcus 的细胞丰度与附近地点的流式细胞仪数据进行比较,结果几乎相同(斜率 = 1.01;Pearson's r = 0.9942)。我们的研究结果表明,这种扩增子测序方案与基因组内部标准相结合,可以准确测量复杂野外样本中海洋微囊藻的绝对细胞数。推而广之,我们希望这种方法能合理估计这些样本中其他扩增类群的体积基因拷贝数。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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