Improved Assembly of Metagenome-Assembled Genomes and Viruses in Tibetan Saline Lake Sediment by HiFi Metagenomic Sequencing

Tao, Ye; Fan, Xiaohui; Zhao, Cheng; Mao, Zhendu; Li, Biao; Xing, Peng; Wu, Qinglong L.

doi:10.1128/spectrum.03328-22

Cited by 15 publications

(13 citation statements)

References 80 publications

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“…These differences likely reflect the inherent genomic characteristics of the groups enriched within each sequencing technology [ 46 ]. Biases in low and high GC content spaces are recognized for SR technologies [ 47 ], while reports on LR approaches have noted anywhere from minimal GC biases in mock communities [ 8 ] to a higher recovery of high GC content sequences in metagenomes [ 46 , 48 , 49 ]. According to our results, most SR-only species belonged to Bacteroidia , Alphaproteobacteria , and Gammaproteobacteria , whereas for LR-only species, Acidiimicrobiia , and Verrucomicrobiae were the two major classes.…”

Section: Resultsmentioning

confidence: 99%

Comparing genomes recovered from time-series metagenomes using long- and short-read sequencing technologies

et al. 2023

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Background Over the past years, sequencing technologies have expanded our ability to examine novel microbial metabolisms and diversity previously obscured by isolation approaches. Long-read sequencing promises to revolutionize the metagenomic field and recover less fragmented genomes from environmental samples. Nonetheless, how to best benefit from long-read sequencing and whether long-read sequencing can provide recovered genomes of similar characteristics as short-read approaches remains unclear. Results We recovered metagenome-assembled genomes (MAGs) from the free-living fraction at four-time points during a spring bloom in the North Sea. The taxonomic composition of all MAGs recovered was comparable between technologies. However, differences consisted of higher sequencing depth for contigs and higher genome population diversity in short-read compared to long-read metagenomes. When pairing population genomes recovered from both sequencing approaches that shared ≥ 99% average nucleotide identity, long-read MAGs were composed of fewer contigs, a higher N50, and a higher number of predicted genes when compared to short-read MAGs. Moreover, 88% of the total long-read MAGs carried a 16S rRNA gene compared to only 23% of MAGs recovered from short-read metagenomes. Relative abundances for population genomes recovered using both technologies were similar, although disagreements were observed for high and low GC content MAGs. Conclusions Our results highlight that short-read technologies recovered more MAGs and a higher number of species than long-read due to an overall higher sequencing depth. Long-read samples produced higher quality MAGs and similar species composition compared to short-read sequencing. Differences in the GC content recovered by each sequencing technology resulted in divergences in the diversity recovered and relative abundance of MAGs within the GC content boundaries.

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Section: Resultsmentioning

confidence: 99%

Comparing genomes recovered from time-series metagenomes using long- and short-read sequencing technologies

et al. 2023

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“…Hybrid assembly approaches, leveraging complementary beneficial attributes of both LR and SR platforms to overcome their limitations, are already being used to study microbial communities in various evniromenments [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] . Assembly of LRs offers a major advantage over SR data alone, due to the ability to achieve greater contiguity, however, it comes at a significant cost due to lower accuracy 1,5,8,11,14,17,[21][22][23][24]32 .…”

Section: Correction and Polishing Affect Gene-and Genome-centric Comm...mentioning

confidence: 99%

“…Though the increasing variety of high-throughput short-and long-read (meta)genomic sequencing technologies are only within their first decades of existence, both the sequencing technologies and software development have flourished and has already been implemented to study microbial ecosystems [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] . Integrating both short-and long-read platforms for single microorganisms or microbial communities is gaining popularity because they compensate for the…”

Section: Background/introductionmentioning

confidence: 99%

“…While there are advanced statistics, both of gene length distributions and read recruitment profiles are relatively simple to calculate, and often precede downstream analyses. In several cases, gene fragmentation and read recruitment profiles, often in comparison to references, have been used to asses recovered MAG quality or the accuracy of assemblies themselves 4,8,[11][12][13]15,17,18,22,45 , but in many cases there was no apparent evaluation or optimization of hybrid assembly of microbial community metagenomes 1,2,5,[9][10][11]14,45 . Given the challenges of applying hybrid assembly approaches for microbial community metagenomes, and the likelihood that no universal approach works best for all datasets, characteristics that are reference-independent and relative to the dataset may be the most suitable to estimate quality and empirically guide optimization.…”

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confidence: 99%

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Simple, reference-independent analyses help optimize hybrid assembly of microbial community metagenomes

Smith,

van Alen,

van Kessel

et al. 2023

Preprint

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Hybrid metagenomic assembly, leveraging both long- and short-read sequencing technologies, of microbial communities is becoming an increasingly accessible approach yet offers challenges that existing benchmarks and pipelines do not comprehensively handle well. One specific methodological knowledge gap is selecting the ideal number of iterations of long read correction and/or short read polishing, and in particular which the features of an assembly to examine to empirically determine them. To account for biological and technological variation, in this study the microbial communities of two laboratory-scale bioreactors were sequenced with both short and long read platforms and assembled with two different, commonly used software packages. Following assembly, long read error correction was iterated ten times, with subsequent ten rounds of short read polishing after common (0 and 2) and extensive (5 and 10) correction iterations. Several assembly characteristics were tracked for each assembly and iteration of correction and polishing: gene fragmentation, short read recruitment, automated binning yields, as well as observed beta-diversity. While long read correction was necessary, the greatest improvements occurred within the first few iterations of short read polishing, and extensive correction and polishing did not tangibly improve assembly quality. Furthermore, essentially all tracked characteristics followed the same patterns; simpler statistics can serve as decent proxies for more complex analyses to save computational resources assessing assembly quality. Hybrid sequencing approaches will likely remain relevant due to the low costs of short read sequencing, therefore it is imperative users are equipped to estimate assembly quality prior to downstream gene- and genome-centric analyses.

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“…While originally limited by high sequence error rates, novel strategies such as the PacBio HiFi sequencing enable the recovery of sequence qualities comparable to short reads (9). PacBio HiFi has recently been applied to various sample types, including human and sheep faecal samples (10, 11), chicken intestinal samples (12), anaerobic digesters (13), seawater (14), and saline lake sediments (15). However, the costs per data unit for long-read technologies are still several orders of magnitude higher than short-read technologies, which has hampered their widespread adoption.…”

Section: Introductionmentioning

confidence: 99%

A comparison of short-read, HiFi long-read, and hybrid strategies for genome-resolved metagenomics

Eisenhofer,

Nesme,

Santos-Bay

et al. 2023

Preprint

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Shotgun metagenomics enables the reconstruction of complex microbial communities at a high level of detail. Such an approach can be conducted using both short-read and long-read sequencing data, as well as a combination of both. To assess the pros and cons of these different approaches, we used 22 faecal DNA extracts collected weekly for 11 weeks from two respective lab mice to study seven performance metrics over four combinations of sequencing depth and technology: i) 20 Gbp of Illumina short-read data, ii) 40 Gbp of short-read data, iii) 20 Gbp of PacBio HiFi long-read data, and iv) 40 Gbp of hybrid (20 Gbp of short-read + 20 Gbp of long-read) data. No strategy was best for all metrics, but instead, each one excelled across different metrics. The long-read approach yielded the best assembly statistics, with the highest N50 and lowest number of contigs. The 40 Gbp short-read approach yielded the highest number of refined bins. Finally, the hybrid approach yielded the longest assemblies, and the highest mapping rate to the bacterial genomes. Our results suggest that while long-read sequencing significantly improves the quality of reconstructed bacterial genomes, it is more expensive and requires deeper sequencing than short-read approaches to recover a comparable amount of reconstructed genomes. The most optimal strategy is study-specific, and depends on how researchers assess the tradeoff between the quantity and quality of recovered genomes.ImportanceOur understanding of microbial communities is limited by the technologies we employ. Here, we test several different DNA sequencing techniques to better understand the pros and cons of each. Long read DNA sequencing allowed for the reconstruction of higher quality and even complete microbial genomes, however, the cost was greater than commonly used short-read DNA sequencing. We suggest researchers consider the trade-offs between each method and decide based on the goals of their research question/s.

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Improved Assembly of Metagenome-Assembled Genomes and Viruses in Tibetan Saline Lake Sediment by HiFi Metagenomic Sequencing

Cited by 15 publications

References 80 publications

Comparing genomes recovered from time-series metagenomes using long- and short-read sequencing technologies

Comparing genomes recovered from time-series metagenomes using long- and short-read sequencing technologies

Simple, reference-independent analyses help optimize hybrid assembly of microbial community metagenomes

A comparison of short-read, HiFi long-read, and hybrid strategies for genome-resolved metagenomics

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