Illumina complete long read assay yields contiguous bacterial genomes from human gut metagenomes
ABSTRACT 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 sequen...
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| Language: | English |
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American Society for Microbiology
2025-08-01
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| Series: | mSystems |
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| Online Access: | https://journals.asm.org/doi/10.1128/msystems.01531-24 |
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| author | Dylan G. Maghini Yuya Kiguchi Aaron E. Darling Leigh G. Monahan Aaron L. Halpern Catherine M. Burke Erich Jaeger Aaron Statham Tiffany Truong Kevin Ying Stephen P. Bruinsma Gary P. Schroth Ami S. Bhatt |
| author_facet | Dylan G. Maghini Yuya Kiguchi Aaron E. Darling Leigh G. Monahan Aaron L. Halpern Catherine M. Burke Erich Jaeger Aaron Statham Tiffany Truong Kevin Ying Stephen P. Bruinsma Gary P. Schroth Ami S. Bhatt |
| author_sort | Dylan G. Maghini |
| collection | DOAJ |
| description | ABSTRACT 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; P = 0.002), and comparable N50 to nanopore assemblies (91.0 ± 43.8 kilobases; P = 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%; P ≤ 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; P ≤ 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. |
| format | Article |
| id | doaj-art-2ec02b0b321e4f7c93f3da7865ecf8e3 |
| institution | Kabale University |
| issn | 2379-5077 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | American Society for Microbiology |
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| series | mSystems |
| spelling | doaj-art-2ec02b0b321e4f7c93f3da7865ecf8e32025-08-20T04:03:26ZengAmerican Society for MicrobiologymSystems2379-50772025-08-0110810.1128/msystems.01531-24Illumina complete long read assay yields contiguous bacterial genomes from human gut metagenomesDylan G. Maghini0Yuya Kiguchi1Aaron E. Darling2Leigh G. Monahan3Aaron L. Halpern4Catherine M. Burke5Erich Jaeger6Aaron Statham7Tiffany Truong8Kevin Ying9Stephen P. Bruinsma10Gary P. Schroth11Ami S. Bhatt12Department of Medicine (Hematology), Stanford University, Stanford, California, USADepartment of Medicine (Hematology), Stanford University, Stanford, California, USAIllumina Inc, San Diego, California, USAIllumina Inc, San Diego, California, USAIllumina Inc, San Diego, California, USAUniversity of Technology Sydney, Ultimo, New South Wales, AustraliaIllumina Inc, San Diego, California, USAIllumina Inc, San Diego, California, USAIllumina Inc, San Diego, California, USAIllumina Inc, San Diego, California, USAIllumina Inc, San Diego, California, USAIllumina Inc, San Diego, California, USADepartment of Medicine (Hematology, Blood and Marrow Transplantation), Stanford University, Stanford, California, USAABSTRACT 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; P = 0.002), and comparable N50 to nanopore assemblies (91.0 ± 43.8 kilobases; P = 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%; P ≤ 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; P ≤ 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.https://journals.asm.org/doi/10.1128/msystems.01531-24metagenomicssequencingmicrobiomegenomics |
| spellingShingle | Dylan G. Maghini Yuya Kiguchi Aaron E. Darling Leigh G. Monahan Aaron L. Halpern Catherine M. Burke Erich Jaeger Aaron Statham Tiffany Truong Kevin Ying Stephen P. Bruinsma Gary P. Schroth Ami S. Bhatt Illumina complete long read assay yields contiguous bacterial genomes from human gut metagenomes mSystems metagenomics sequencing microbiome genomics |
| title | Illumina complete long read assay yields contiguous bacterial genomes from human gut metagenomes |
| title_full | Illumina complete long read assay yields contiguous bacterial genomes from human gut metagenomes |
| title_fullStr | Illumina complete long read assay yields contiguous bacterial genomes from human gut metagenomes |
| title_full_unstemmed | Illumina complete long read assay yields contiguous bacterial genomes from human gut metagenomes |
| title_short | Illumina complete long read assay yields contiguous bacterial genomes from human gut metagenomes |
| title_sort | illumina complete long read assay yields contiguous bacterial genomes from human gut metagenomes |
| topic | metagenomics sequencing microbiome genomics |
| url | https://journals.asm.org/doi/10.1128/msystems.01531-24 |
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