Maghini Dylan G, Kiguchi Yuya, Darling Aaron E, Monahan Leigh G, Halpern Aaron L, Burke Catherine M, Jaeger Erich, Statham Aaron, Truong Tiffany, Ying Kevin, Bruinsma Stephen P, Schroth Gary P, Bhatt Ami S
Department of Medicine (Hematology), Stanford University, Stanford, California, USA.
Illumina Inc, San Diego, California, USA.
mSystems. 2025 Jul 23:e0153124. doi: 10.1128/msystems.01531-24.
Metagenomics enables direct investigation of the gene content and potential functions of gut bacteria without isolation and culture. However, metagenome-assembled genomes are often incomplete and have low contiguity due to challenges in assembling repeated genomic elements. Long-read sequencing approaches have successfully yielded circular bacterial genomes directly from metagenomes, but these approaches require high DNA input and can have high error rates. Illumina has recently launched the Illumina Complete Long Read (ICLR) assay, a new approach for generating kilobase-scale reads with low DNA input requirements and high accuracy. Here, we evaluate the performance of ICLR sequencing for gut metagenomics for the first time. We sequenced a microbial mock community and 10 human gut microbiome samples with standard, shotgun 2 × 150 paired-end sequencing, ICLR sequencing, and nanopore long-read sequencing and compared performance in read lengths, assembly contiguity, and bin quality. We find that ICLR human metagenomic assemblies have higher N50 (119.5 ± 24.8 kilobases) than short read assemblies (9.9 ± 4.5 kilobases; = 0.002), and comparable N50 to nanopore assemblies (91.0 ± 43.8 kilobases; = 0.32). Additionally, we find that ICLR draft microbial genomes are more complete (94.0% ± 20.6%) than nanopore draft genomes (85.9% ± 23.0%; 0.001), and that nanopore draft genomes have truncated gene lengths (924.6 ± 114.7 base pairs) relative to ICLR genomes (954.6 ± 71.5 base pairs; 0.001). Overall, we find that ICLR sequencing is a promising method for the accurate assembly of microbial genomes from gut metagenomes.IMPORTANCEMetagenomic sequencing allows scientists to directly measure the genome content and structure of microbes residing in complex microbial communities. Traditional short-read metagenomic sequencing methods often yield fragmented genomes, whereas advanced long-read sequencing methods improve genome assembly quality but often suffer from high error rates and are logistically limited due to high input requirements. A new method, the Illumina Complete Long Read (ICLR) assay, is capable of generating highly accurate kilobase-scale sequencing reads with minimal input material. To evaluate the utility of ICLR in metagenomic contexts, we applied short-read, long-read, and ICLR methods to simple and complex microbial communities. We found that ICLR outperforms short-read methods and yields comparable metagenomic assemblies to standard long-read approaches while requiring less input material. Overall, ICLR represents an additional option for assembling complete genomes from complex metagenomes.
宏基因组学能够直接研究肠道细菌的基因组成和潜在功能，而无需进行分离和培养。然而，由于在组装重复基因组元件方面存在挑战，宏基因组组装的基因组通常不完整且连续性较低。长读长测序方法已成功地直接从宏基因组中获得环状细菌基因组，但这些方法需要大量的DNA输入，并且可能具有较高的错误率。Illumina公司最近推出了Illumina Complete Long Read（ICLR）检测方法，这是一种新的方法，能够以低DNA输入要求和高精度生成千碱基规模的读长。在这里，我们首次评估了ICLR测序在肠道宏基因组学中的性能。我们使用标准的鸟枪法2×150双端测序、ICLR测序和纳米孔长读长测序对一个微生物模拟群落和10个人类肠道微生物组样本进行了测序，并比较了读长、组装连续性和分箱质量方面的性能。我们发现，ICLR人类宏基因组组装的N50（119.5±24.8千碱基）高于短读长组装（9.9±4.5千碱基；P = 0.002），并且与纳米孔组装的N50相当（91.0±43.8千碱基；P = 0.32）。此外，我们发现ICLR微生物基因组草图比纳米孔基因组草图更完整（94.0%±20.6%）（85.9%±23.0%；P ＜0.001），并且纳米孔基因组草图的基因长度（924.6±114.7碱基对）相对于ICLR基因组（954.6±71.5碱基对；P ＜0.001）有所截断。总体而言，我们发现ICLR测序是一种从肠道宏基因组中准确组装微生物基因组的有前途的方法。
重要性
宏基因组测序使科学家能够直接测量复杂微生物群落中微生物的基因组内容和结构。传统的短读长宏基因组测序方法通常会产生碎片化的基因组，而先进的长读长测序方法提高了基因组组装质量，但往往存在高错误率，并且由于高输入要求在后勤方面受到限制。一种新的方法，即Illumina Complete Long Read（ICLR）检测方法，能够以最少的输入材料生成高度准确的千碱基规模测序读长。为了评估ICLR在宏基因组学背景下的实用性，我们将短读长、长读长和ICLR方法应用于简单和复杂的微生物群落。我们发现ICLR优于短读长方法，并且在需要更少输入材料的情况下，产生的宏基因组组装与标准长读长方法相当。总体而言，ICLR是从复杂宏基因组中组装完整基因组的另一种选择。
