A novel ultra high-throughput 16S rRNA gene amplicon sequencing library preparation method for the Illumina HiSeq platform

AbstractBackgroundAdvances in sequencing technologies and bioinformatics have made the analysis of microbial communities almost routine. Nonetheless, the need remains to improve on the techniques used for gathering such data, including increasing throughput while lowering cost, and benchmarking the techniques so that potential sources of bias can be better characterized.ResultsWe present a triple-index amplicon sequencing strategy that uses a two-stage PCR protocol. The strategy was extensively benchmarked through analysis of a mock community in order to assess biases introduced by sample indexing, number of PCR cycles, and template concentration. We further evaluated the method through re-sequencing of a standardized environmental sample. Finally, we evaluated our protocol on a set of fecal samples from a small cohort of healthy adults, demonstrating good performance in a realistic experimental setting. Between-sample variation was mainly related to batch effects, such as DNA extraction, while sample indexing was also a significant source of bias. PCR cycle number strongly influenced chimera formation and affected relative abundance estimates of species with high GC content. Libraries were sequenced using the Illumina HiSeq and MiSeq platforms to demonstrate that this protocol is highly scalable to sequence thousands of samples at a very low cost.ConclusionsHere, we provide the most comprehensive study of performance and bias inherent to a 16S rRNA gene amplicon sequencing method to date. Triple-indexing greatly reduces the number of long custom DNA oligos required for library preparation, while the inclusion of variable length heterogeneity spacers minimizes the need for PhiX spike-in. This design results in a significant cost reduction of highly multiplexed amplicon sequencing. The biases we characterize highlight the need for highly standardized protocols. Reassuringly, we find that the biological signal is a far stronger structuring factor than the various sources of bias.

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Next-Generation Sequencing Library Preparation for 16S rRNA Microbiome Analysis After Serpin Treatment

Methods in Molecular Biology - Serpins ◽

10.1007/978-1-4939-8645-3_14 ◽

2018 ◽

pp. 213-221 ◽

Cited By ~ 1

Author(s):

Juan Maldonado ◽

Jordan R. Yaron ◽

Liqiang Zhang ◽

Alexandra Lucas

Keyword(s):

Next Generation Sequencing ◽

16S Rrna ◽

Library Preparation ◽

Next Generation ◽

Microbiome Analysis ◽

Sequencing Library ◽

Sequencing Library Preparation ◽

Generation Sequencing

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Comparing library preparation methods for SARS-CoV-2 multiplex amplicon sequencing on the Illumina MiSeq platform

10.1101/2020.06.16.154286 ◽

2020 ◽

Cited By ~ 2

Author(s):

Elizabeth M. Batty ◽

Theerarat Kochakarn ◽

Arporn Wangwiwatsin ◽

Khajohn Joonlasak ◽

Angkana T. Huang ◽

...

Keyword(s):

Variant Calling ◽

Illumina Miseq ◽

Amplicon Sequencing ◽

Library Preparation ◽

Preparation Methods ◽

Base Calling ◽

Sequencing Library ◽

Miseq Platform ◽

Downstream Analysis ◽

Sequencing Library Preparation

AbstractGenomic surveillance has a key role in tracking the ongoing COVID-19 pandemic, but information on how different sequencing library preparation approaches affect the data produced are lacking. We compared three library preparation methods using both tagmentation (Nextera XT and Nextera Flex) and ligation-based (KAPA HyperPrep) approaches on both positive and negative samples to provide insights into any methodological differences between the methods, and validate their use in SARS-CoV-2 amplicon sequencing. We show that all three library preparation methods allow us to recover near-complete SARS-CoV-2 genomes with identical SNP calls. The Nextera Flex and KAPA library preparation methods gave better coverage than libraries prepared with Nextera XT, which required more reads to call the same number of genomic positions. The KAPA ligation-based approach shows the lowest levels of human contamination, but contaminating reads had no effect on the downstream analysis. We found some examples of library preparation-specific differences in minority variant calling. Overall our data shows that the choice of Illumina library preparation method has minimal effects on consensus base calling and downstream phylogenetic analysis, and suggests that all methods would be suitable for use if specific reagents are difficult to obtain.

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High-Throughput Miniaturized 16S rRNA Amplicon Library Preparation Reduces Costs while Preserving Microbiome Integrity

mSystems ◽

10.1128/msystems.00166-18 ◽

2018 ◽

Vol 3 (6) ◽

Cited By ~ 17

Author(s):

Jeremiah J. Minich ◽

Greg Humphrey ◽

Rodolfo A. S. Benitez ◽

Jon Sanders ◽

Austin Swafford ◽

...

Keyword(s):

16S Rrna ◽

Microbial Ecology ◽

High Throughput ◽

Preparation Method ◽

Library Preparation ◽

Reaction Volume ◽

Liquid Handling ◽

Amplicon Library ◽

The Cost ◽

Library Preparation Method

ABSTRACT Next-generation sequencing technologies have enabled many advances across biology, with microbial ecology benefiting primarily through expanded sample sizes. Although the cost of running sequencing instruments has decreased substantially over time, the price of library preparation methods has largely remained unchanged. In this study, we developed a low-cost miniaturized (5-µl volume) high-throughput (384-sample) amplicon library preparation method with the Echo 550 acoustic liquid handler. Our method reduces costs of library preparation to $1.42 per sample, a 58% reduction compared to existing automated methods and a 21-fold reduction from commercial kits, without compromising sequencing success or distorting the microbial community composition analysis. We further validated the optimized method by sampling five body sites from 46 Pacific chub mackerel fish caught across 16 sampling events over seven months from the Scripps Institution of Oceanography pier in La Jolla, CA. Fish microbiome samples were processed with the miniaturized 5-µl reaction volume with 0.2 µl of genomic DNA (gDNA) and the standard 25-µl reaction volume with 1 µl of gDNA. Between the two methods, alpha diversity was highly correlated (R2 > 0.95), while distances of technical replicates were much lower than within-body-site variation (P < 0.0001), further validating the method. The cost savings of implementing the miniaturized library preparation (going from triplicate 25-µl reactions to triplicate 5-µl reactions) are large enough to cover a MiSeq sequencing run for 768 samples while preserving accurate microbiome measurements. IMPORTANCE Reduced costs of sequencing have tremendously impacted the field of microbial ecology, allowing scientists to design more studies with larger sample sizes that often exceed 10,000 samples. Library preparation costs have not kept pace with sequencing prices, although automated liquid handling robots provide a unique opportunity to bridge this gap while also decreasing human error. Here, we take advantage of an acoustic liquid handling robot to develop a high-throughput miniaturized library preparation method of a highly cited and broadly used 16S rRNA gene amplicon reaction. We evaluate the potential negative effects of reducing the PCR volume along with varying the amount of gDNA going into the reaction. Our optimized method reduces sample-processing costs while continuing to generate a high-quality microbiome readout that is indistinguishable from the original method.

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Homology-based enzymatic DNA fragment assembly-based illumina sequencing library preparation

Biology Methods and Protocols ◽

10.1093/biomethods/bpy001 ◽

2018 ◽

Vol 3 (1) ◽

Author(s):

Hiroshi Shinozuka ◽

Shimna Sudheesh ◽

Maiko Shinozuka ◽

Noel O I Cogan

Keyword(s):

Single Molecule ◽

Dna Assembly ◽

Nucleotide Polymorphisms ◽

Library Preparation ◽

Illumina Hiseq ◽

Fragment Assembly ◽

Sequencing Library ◽

Dna Fragment ◽

Sequencing Library Preparation ◽

Dna Fragment Assembly

Abstract The current Illumina HiSeq and MiSeq platforms can generate paired-end reads of up to 2 x 250 bp and 2 x 300 bp in length, respectively. These read lengths may be substantially longer than genomic regions of interest when a DNA sequencing library is prepared through a target enrichment-based approach. A sequencing library preparation method has been developed based on the homology-based enzymatic DNA fragment assembly scheme to allow processing of multiple PCR products within a single read. Target sequences were amplified using locus-specific PCR primers with 8 bp tags, and using the tags, homology-based enzymatic DNA assembly was performed with DNA polymerase, T7 exonuclease and T4 DNA ligase. Short PCR amplicons can hence be assembled into a single molecule, along with sequencing adapters specific to the Illumina platforms. As a proof-of-concept experiment, short PCR amplicons (57–66 bp in length) derived from genomic DNA templates of field pea and containing variable nucleotide locations were assembled and sequenced on the MiSeq platform. The results were validated with other genotyping methods. When 5 PCR amplicons were assembled, 4.3 targeted sequences (single-nucleotide polymorphisms) on average were successfully identified within each read. The utility of this for sequencing of short fragments has consequently been demonstrated.

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Much ado about nothing? Off-target amplification can lead to false-positive bacterial brain microbiome detection in healthy and Parkinson’s disease individuals

Microbiome ◽

10.1186/s40168-021-01012-1 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Janis R. Bedarf ◽

Naiara Beraza ◽

Hassan Khazneh ◽

Ezgi Özkurt ◽

David Baker ◽

...

Keyword(s):

Parkinson’S Disease ◽

16S Rrna ◽

16S Rrna Gene ◽

False Positive ◽

Gene Sequencing ◽

Bacterial Biomass ◽

16S Rrna Gene Sequencing ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Rrna Gene Sequencing

Abstract Background Recent studies suggested the existence of (poly-)microbial infections in human brains. These have been described either as putative pathogens linked to the neuro-inflammatory changes seen in Parkinson’s disease (PD) and Alzheimer’s disease (AD) or as a “brain microbiome” in the context of healthy patients’ brain samples. Methods Using 16S rRNA gene sequencing, we tested the hypothesis that there is a bacterial brain microbiome. We evaluated brain samples from healthy human subjects and individuals suffering from PD (olfactory bulb and pre-frontal cortex), as well as murine brains. In line with state-of-the-art recommendations, we included several negative and positive controls in our analysis and estimated total bacterial biomass by 16S rRNA gene qPCR. Results Amplicon sequencing did detect bacterial signals in both human and murine samples, but estimated bacterial biomass was extremely low in all samples. Stringent reanalyses implied bacterial signals being explained by a combination of exogenous DNA contamination (54.8%) and false positive amplification of host DNA (34.2%, off-target amplicons). Several seemingly brain-enriched microbes in our dataset turned out to be false-positive signals upon closer examination. We identified off-target amplification as a major confounding factor in low-bacterial/high-host-DNA scenarios. These amplified human or mouse DNA sequences were clustered and falsely assigned to bacterial taxa in the majority of tested amplicon sequencing pipelines. Off-target amplicons seemed to be related to the tissue’s sterility and could also be found in independent brain 16S rRNA gene sequences. Conclusions Taxonomic signals obtained from (extremely) low biomass samples by 16S rRNA gene sequencing must be scrutinized closely to exclude the possibility of off-target amplifications, amplicons that can only appear enriched in biological samples, but are sometimes assigned to bacterial taxa. Sequences must be explicitly matched against any possible background genomes present in large quantities (i.e., the host genome). Using close scrutiny in our approach, we find no evidence supporting the hypothetical presence of either a brain microbiome or a bacterial infection in PD brains.

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Methyltransferase-directed orthogonal tagging and sequencing of miRNAs and bacterial small RNAs

BMC Biology ◽

10.1186/s12915-021-01053-w ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Milda Mickutė ◽

Kotryna Kvederavičiūtė ◽

Aleksandr Osipenko ◽

Raminta Mineikaitė ◽

Saulius Klimašauskas ◽

...

Keyword(s):

Rna Sequencing ◽

Regulatory Networks ◽

Library Preparation ◽

Rna Seq ◽

Basic Principles ◽

Cofactor Binding ◽

Sequencing Library ◽

Sequencing Library Preparation ◽

Target Rna

Abstract Background Targeted installation of designer chemical moieties on biopolymers provides an orthogonal means for their visualisation, manipulation and sequence analysis. Although high-throughput RNA sequencing is a widely used method for transcriptome analysis, certain steps, such as 3′ adapter ligation in strand-specific RNA sequencing, remain challenging due to structure- and sequence-related biases introduced by RNA ligases, leading to misrepresentation of particular RNA species. Here, we remedy this limitation by adapting two RNA 2′-O-methyltransferases from the Hen1 family for orthogonal chemo-enzymatic click tethering of a 3′ sequencing adapter that supports cDNA production by reverse transcription of the tagged RNA. Results We showed that the ssRNA-specific DmHen1 and dsRNA-specific AtHEN1 can be used to efficiently append an oligonucleotide adapter to the 3′ end of target RNA for sequencing library preparation. Using this new chemo-enzymatic approach, we identified miRNAs and prokaryotic small non-coding sRNAs in probiotic Lactobacillus casei BL23. We found that compared to a reference conventional RNA library preparation, methyltransferase-Directed Orthogonal Tagging and RNA sequencing, mDOT-seq, avoids misdetection of unspecific highly-structured RNA species, thus providing better accuracy in identifying the groups of transcripts analysed. Our results suggest that mDOT-seq has the potential to advance analysis of eukaryotic and prokaryotic ssRNAs. Conclusions Our findings provide a valuable resource for studies of the RNA-centred regulatory networks in Lactobacilli and pave the way to developing novel transcriptome and epitranscriptome profiling approaches in vitro and inside living cells. As RNA methyltransferases share the structure of the AdoMet-binding domain and several specific cofactor binding features, the basic principles of our approach could be easily translated to other AdoMet-dependent enzymes for the development of modification-specific RNA-seq techniques.

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Meta-Apo improves accuracy of 16S-amplicon-based prediction of microbiome function

BMC Genomics ◽

10.1186/s12864-020-07307-1 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Gongchao Jing ◽

Yufeng Zhang ◽

Wenzhi Cui ◽

Lu Liu ◽

Jian Xu ◽

...

Keyword(s):

16S Rrna ◽

Large Scale ◽

Low Cost ◽

Human Microbiome ◽

Amplicon Sequencing ◽

Training Sample ◽

Rrna Gene ◽

16S Amplicon Sequencing ◽

Cross Platform ◽

Functional Profiles

Abstract Background Due to their much lower costs in experiment and computation than metagenomic whole-genome sequencing (WGS), 16S rRNA gene amplicons have been widely used for predicting the functional profiles of microbiome, via software tools such as PICRUSt 2. However, due to the potential PCR bias and gene profile variation among phylogenetically related genomes, functional profiles predicted from 16S amplicons may deviate from WGS-derived ones, resulting in misleading results. Results Here we present Meta-Apo, which greatly reduces or even eliminates such deviation, thus deduces much more consistent diversity patterns between the two approaches. Tests of Meta-Apo on > 5000 16S-rRNA amplicon human microbiome samples from 4 body sites showed the deviation between the two strategies is significantly reduced by using only 15 WGS-amplicon training sample pairs. Moreover, Meta-Apo enables cross-platform functional comparison between WGS and amplicon samples, thus greatly improve 16S-based microbiome diagnosis, e.g. accuracy of gingivitis diagnosis via 16S-derived functional profiles was elevated from 65 to 95% by WGS-based classification. Therefore, with the low cost of 16S-amplicon sequencing, Meta-Apo can produce a reliable, high-resolution view of microbiome function equivalent to that offered by shotgun WGS. Conclusions This suggests that large-scale, function-oriented microbiome sequencing projects can probably benefit from the lower cost of 16S-amplicon strategy, without sacrificing the precision in functional reconstruction that otherwise requires WGS. An optimized C++ implementation of Meta-Apo is available on GitHub (https://github.com/qibebt-bioinfo/meta-apo) under a GNU GPL license. It takes the functional profiles of a few paired WGS:16S-amplicon samples as training, and outputs the calibrated functional profiles for the much larger number of 16S-amplicon samples.

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Handling of spurious sequences affects the outcome of high-throughput 16S rRNA gene amplicon profiling

ISME Communications ◽

10.1038/s43705-021-00033-z ◽

2021 ◽

Vol 1 (1) ◽

Author(s):

Sandra Reitmeier ◽

Thomas C. A. Hitch ◽

Nicole Treichel ◽

Nikolaos Fikas ◽

Bela Hausmann ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Diversity Analysis ◽

Careful Attention ◽

Operational Taxonomic Units ◽

Basic Concepts ◽

Gnotobiotic Mice ◽

Mock Communities

Abstract16S rRNA gene amplicon sequencing is a popular approach for studying microbiomes. However, some basic concepts have still not been investigated comprehensively. We studied the occurrence of spurious sequences using defined microbial communities based on data either from the literature or generated in three sequencing facilities and analyzed via both operational taxonomic units (OTUs) and amplicon sequence variants (ASVs) approaches. OTU clustering and singleton removal, a commonly used approach, delivered approximately 50% (mock communities) to 80% (gnotobiotic mice) spurious taxa. The fraction of spurious taxa was generally lower based on ASV analysis, but varied depending on the gene region targeted and the barcoding system used. A relative abundance of 0.25% was found as an effective threshold below which the analysis of spurious taxa can be prevented to a large extent in both OTU- and ASV-based analysis approaches. Using this cutoff improved the reproducibility of analysis, i.e., variation in richness estimates was reduced by 38% compared with singleton filtering using six human fecal samples across seven sequencing runs. Beta-diversity analysis of human fecal communities was markedly affected by both the filtering strategy and the type of phylogenetic distances used for comparison, highlighting the importance of carefully analyzing data before drawing conclusions on microbiome changes. In summary, handling of artifact sequences during bioinformatic processing of 16S rRNA gene amplicon data requires careful attention to avoid the generation of misleading findings. We propose the concept of effective richness to facilitate the comparison of alpha-diversity across studies.

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