Reducing amplification artifacts in high multiplex amplicon sequencing by using molecular barcodes

Background

PCR amplicon sequencing has been widely used as a targeted approach for both DNA and RNA sequence analysis. High multiplex PCR has further enabled the enrichment of hundreds of amplicons in one simple reaction. At the same time, the performance of PCR amplicon sequencing can be negatively affected by issues such as high duplicate reads, polymerase artifacts and PCR amplification bias. Recently researchers have made some good progress in addressing these shortcomings by incorporating molecular barcodes into PCR primer design. So far, most work has been demonstrated using one to a few pairs of primers, which limits the size of the region one can analyze.

Results

We developed a simple protocol, which enables the use of molecular barcodes in high multiplex PCR with hundreds of amplicons. Using this protocol and reference materials, we demonstrated the applications in accurate variant calling at very low fraction over a large region and in targeted RNA quantification. We also evaluated the protocol’s utility in profiling FFPE samples.

Conclusions

We demonstrated the successful implementation of molecular barcodes in high multiplex PCR, with multiplex scale many times higher than earlier work. We showed that the new protocol combines the benefits of both high multiplex PCR and molecular barcodes, i.e. the analysis of a very large region, low DNA input requirement, very good reproducibility and the ability to detect as low as 1 % mutations with minimal false positives (FP).

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1806-8) contains supplementary material, which is available to authorized users.     

Bacterial diversity in two coastal lagoons deduced from 16S rDNA PCR amplification and partial sequencing

Abstract: Amplification and sequence analysis of the 16S rRNA genes from DNA samples extracted directly from the environment allows the study of microbial diversity in natural ecosystems without the need for cultivation. In this study this methodology has been applied to two coastal lagoons. Activity and numbers of heterotrophic bacteria have indicated that, as expected, Prévost lagoon (located on the French Mediterranean coast) is more eutrophic than that of the Arcachon Bay (French Atlantic coast). Analysis of partial 16S rRNA gene sequences revealed that, in both environments, a relatively large number of clones related to Cytophaga/Flexibacter/Bacteroides as well as to α-Proteobacteria were found. One hundred percent similarity with the sequences of the data bases were not found for any of the more than a hundred clones studied, in fact for most clones maximum similarity was below 95% for the approx. 200 bases sequenced. Similarity was not higher with any of the sequences found for the 14 isolates (pure cultures) obtained from the same samples. Redundancy, i.e. number of identical sequences, was higher in the samples from Arcachon. In addition, sequences related to representatives of ten major phylogenetic branches of Bacteria were obtained from Prévost lagoon; however only five branches were represented by the data from Arcachon. These findings indicated a higher bacterial phylogenetic diversity in the Prévost lagoon.     

PCR amplification of 16S rDNA from lyophilized cell cultures facilitates studies in molecular systematics

The sequence of the major portion of aBacillus cycloheptanicus strain SCH^T 16S rRNA gene is reported. This sequence suggests thatB. cycloheptanicus is genetically quite distinct from traditionalBacillus strains (e.g.,B. subtilis) and may be properly regarded as belonging to a different genus. The sequence was determined from DNA that was produced by direct amplification of ribosomal DNA from a lyophilized cell pellet with straightforward polymerase chain reaction (PCR) procedures. By obviating the need to revive cell cultures from the lyophile pellet, this approach facilitates rapid 16S rDNA sequencing and thereby advances studies in molecular systematics.The EMBL accession number for the 16S rRNA sequence is X51928.     

PCR反应条件对16S rRNA基因扩增子测序准确性的影响

【背景】16S rRNA基因扩增子测序技术是一种不依赖培养而获得样本中细菌种群结构、相对丰度等信息的方法。高通量测序技术实验步骤较多,每一步骤细微的差别都可能在最终的测序结果中放大,并造成测序结果与实际情况的偏差。【目的】基于MiSeq测序平台,探讨PCR反应体系中扩增引物序列、退火温度、模板起始量、扩增循环数和变性时间等5个因素对16S rRNA基因测序结果的影响。【方法】对mock DNA的16S rRNA基因扩增子进行测序,分别分析不同的扩增引物、退火温度、模板起始量、循环数和变性时间对数据准确性的影响。【结果】不同的扩增引物对检测结果有较大的影响,采用的4组引物中,引物B (V3–V4,341F/806R)的准确性最好,引物A(V3–V4,341F/805R)次之。比较不同退火温度(52、55和60℃)对检测准确性的影响,退火温度60℃的结果最接近理论值。模板起始量(2、10和50 ng)的检测结果显示,mock DNA起始量为2ng的结果准确性最高。相较于其他3组(15+18、25+8和30+8),循环数为(20+8)的检测结果最接近mock DNA的理论值。不同变性时间(3...     

Rapid identification of bacteria by real-time amplification and sequencing of the 16S rRNA gene

To allow rapid identification of bacteria in pure cultures and blood culture bottles, an assay was developed which is based on real-time amplification and sequencing of bacterial 16 S rRNA genes. In principle, this assay allows identification of bacteria from pure cultures within 6.5 h, and from blood cultures within approximately 7 h.     

Influence of DNA extraction and PCR amplification on studies of soil fungal communities based on amplicon sequencing

Most studies involving next-generation amplicon sequencing of microbial communities from environmental studies lack replicates. DNA extraction and PCR effects on the variation of read abundances of operational taxonomic units generated from deep amplicon 454 pyrosequencing was investigated using soil samples from an agricultural field with diseased pea. One sample was extracted four times, and one of these samples was PCR amplified four times to obtain eight replicates in total. Results showed that species richness was consistent among replicates. Variation among dominant taxa was low across replicates, whereas rare operational taxonomic units showed higher variation among replicates. The results indicate that pooling of several extractions and PCR amplicons will decrease variation among samples.     

The PCR amplification of non-tuberculous mycobacterial 16S rRNA sequences from soil

Non-tuberculous mycobacteria are free living saprophytic organisms commonly found in soil and water. Some are major causes of opportunistic infection, particularly in immuno-compromised patients, and may influence the efficacy of bacille Calmette-Guérin vaccinations. Many of these organisms are not amenable to culture, so information about their distribution is limited. PCR primers designed to amplify part of the mycobacterial 16S rRNA gene were applied to DNA extracted from cultured organisms and soil. The PCR products from soil contained sequences with similarity to slow growing mycobacteria similar to Mycobacterium lentiflavum, and to fast growing mycobacteria such as the xenobiotic degraders PYR-I and RJGII.     

Designation of Streptomycete 16S and 23S rRNA-based target regions for oligonucleotide probes

The 16S and 23S rRNA of various Streptomyces species were partially sequenced and screened for the presence of stretches that could define all members of the genus, groups of species, or individual species. Nucleotide 929 (Streptomyces ambofaciens nomenclature [J.L. Pernodet, M.T. Alegre, F. Boccard, and M. Guerineau, Gene 79:33-46, 1989]) is a nucleotide highly unique to Streptomyces species which, in combination with flanking regions, allowed the designation of a genus-specific probe. Regions 158 through 203 of the 16S rRNA and 1518 through 1645 of the 23S rRNA (helix 54 [Pernodet et al., Gene 79:33-46, 1989]) have a high potential to define species, whereas the degree of variation in regions 982 through 998 and 1102 through 1122 of the 16S rRNA is less pronounced but characteristic for at least certain species. Alone or in combination with each other, these regions may serve as target sites for synthetic oligonucleotide probes and primers to be used in the determination of pure cultures and in the characterization of community structures. The specificity of several probes is demonstrated by dot blot hybridization.     

Enhancing PCR amplification and sequencing using DNA-binding proteins

The polymerase chain reaction (PCR) is a powerful core molecular biology technique, which when coupled to chain termination sequencing allows gene and DNA sequence information to be derived rapidly. A number of modifications to the basic PCR format have been developed in an attempt to increase amplification efficiency and the specificity of the reaction. We have applied the use of DNA-binding protein, gene 32 protein from bacteriophage T4 (T4gp32) to increase amplification efficiency with a number of diverse templates. In addition, we have found that using single-stranded DNA-binding protein (SSB) or recA protein in DNA sequencing reactions dramatically increases the resolution of sequencing runs. The use of DNA-binding proteins in amplification and sequencing may prove to be generally applicable in improving the yield and quality of a number of templates from various sources.     

16S rRNA-based analysis of microbiota from the cecum of broiler chickens.

The microbiota of the intestinal tract of chickens plays an important role in inhibiting the establishment of intestinal pathogens. Earlier culturing and microscopic examinations indicated that only a fraction of the bacteria in the cecum of chickens could be grown in the laboratory. Therefore, a survey of cecal bacteria was done by retrieval of 16S rRNA gene sequences from DNA isolated from the cecal content and the cecal mucosa. The ribosomal gene sequences were amplified with universal primers and cloned or subjected to temporal temperature gradient gel electrophoresis (TTGE). Partial 16S rRNA gene sequences were determined from the clones and from the major bands in TTGE gels. A total of 1,656 partial 16S rRNA gene sequences were obtained and compared to sequences in the GenBank. The comparison indicated that 243 different sequences were present in the samples. Overall, sequences representing 50 phylogenetic groups or subgroups of bacteria were found, but approximately 89% of the sequences represented just four phylogenetic groups (Clostridium leptum, Sporomusa sp., Clostridium coccoides, and enterics). Sequences of members of the Bacteroides group, the Bifidobacterium infantis subgroup, and of Pseudomonas sp. each accounted for less than 2% of the total. Sequences related to those from the Escherichia sp. subgroup and from Lactobacillus, Pseudomonas, and Bifidobacterium spp. were generally between 98 and 100% identical to sequences already deposited in the GenBank. Sequences most closely related to those of the other bacteria were generally 97% or less identical to those in the databases and therefore might be from currently unknown species. TTGE and random cloning indicated that certain phylogenetic subgroups were common to all birds analyzed, but sequence data from random cloning also provided evidence for qualitative and quantitative differences among the cecal microbiota of individual birds reared under very similar conditions.     

Designation of Streptomycete 16S and 23S rRNA-based target regions for oligonucleotide probes.

The 16S and 23S rRNA of various Streptomyces species were partially sequenced and screened for the presence of stretches that could define all members of the genus, groups of species, or individual species. Nucleotide 929 (Streptomyces ambofaciens nomenclature [J.L. Pernodet, M.T. Alegre, F. Boccard, and M. Guerineau, Gene 79:33-46, 1989]) is a nucleotide highly unique to Streptomyces species which, in combination with flanking regions, allowed the designation of a genus-specific probe. Regions 158 through 203 of the 16S rRNA and 1518 through 1645 of the 23S rRNA (helix 54 [Pernodet et al., Gene 79:33-46, 1989]) have a high potential to define species, whereas the degree of variation in regions 982 through 998 and 1102 through 1122 of the 16S rRNA is less pronounced but characteristic for at least certain species. Alone or in combination with each other, these regions may serve as target sites for synthetic oligonucleotide probes and primers to be used in the determination of pure cultures and in the characterization of community structures. The specificity of several probes is demonstrated by dot blot hybridization.     

PCR amplification of microalgal DNA for sequencing and species identification: studies on fixatives and algal growth stages

Cultured strains and individually isolated dinoflagellate cells from field samples were preserved in different fixatives to find a method of cell preservation that could provide DNA template in PCR reactions and preserve cell morphology for microscopic studies. Lugol’s solution and various ethanol concentrations all showed shortcomings, whereas an initial formalin preservation step followed by storage in 100% methanol fulfilled both demands. Cells could be stored up to 1 year and still provide functional DNA template for positive PCR reactions. The amplified fragment was approximately 700 bp of the D1/D2 region of the LSU rDNA, which is to our knowledge significantly longer than the low-molecular-weight DNA typically reported from formalin preserved samples. By cloning and sequencing the PCR product and subsequently aligning the sequences with previously sequenced fragments of the same or similar species, we confirmed that no base pair alteration had taken place during the time that the cells were fixed and frozen. In another experiment it was demonstrated that the growth phase of cultured Alexandrium minutum did not have any influence on the result of PCR reactions. This was true for extracted DNA from cultures and for direct PCR with a small number of disrupted cells. Phenol/chlorophorm/isoamylalcohol extraction proved to be an unpredictable method for DNA extraction, whereas direct PCR on isolated cells was more reliable. Extracted DNA purified with a commercial DNA cleaning kit always rendered a positive PCR. The environmental condition for cells to be used as DNA template in PCR is discussed.     

Development of a 23S rRNA-based PCR assay for the identification of Pasteurella multocida

AIMS: The aim of this work was to develop a rapid diagnostic test for Pasteurella multocida. METHODS AND RESULTS: A polymerase chain reaction (PCR) assay using primers derived from the 23S rRNA gene sequence of Past. multocida was developed. The PCR assay correctly identified all 144 isolates of Past. multocida tested, including type strains of the three subspecies as well as the reference strains for the Heddleston and Carter typing schemes. Of 20 closely related bacteria from the family Pasteurellaceae tested, only the type strains of Past. canis biovar 2 and Past. avium biovar 2 were positive. These two bacteria, formerly known as Bisgaard Taxon 13, are the closest phylogenetic relatives of Past. multocida based on 16S ribosomal rRNA. All phylogenetically unrelated avian and porcine organisms tested were negative. CONCLUSION: This PCR enables rapid identification of Past. multocida colonies from avian or porcine origin. SIGNIFICANCE AND IMPACT OF THE STUDY: Veterinary diagnostic laboratories can use this PCR to rapidly and accurately diagnose fowl cholera and porcine pasteurellosis.     

Development and validation of a prototype 16S rRNA-based taxonomic microarray for Alphaproteobacteria

The microarray approach has been proposed for high throughput analysis of the microbial community by providing snapshots of the microbial diversity under different environmental conditions. For this purpose, a prototype of a 16S rRNA-based taxonomic microarray was developed and evaluated for assessing bacterial community diversity. The prototype microarray is composed of 122 probes that target bacteria at various taxonomic levels from phyla to species (mostly Alphaproteobacteria). The prototype microarray was first validated using bacteria in pure culture. Differences in the sequences of probes and potential target DNAs were quantified as weighted mismatches (WMM) in order to evaluate hybridization reliability. As a general feature, probes having a WMM > 2 with target DNA displayed only 2.8% false positives. The prototype microarray was subsequently tested with an environmental sample, which consisted of an Agrobacterium-related polymerase chain reaction amplicon from a maize rhizosphere bacterial community. Microarray results were compared to results obtained by cloning-sequencing with the same DNA. Microarray analysis enabled the detection of all 16S rRNA gene sequences found by cloning-sequencing. Sequences representing only 1.7% of the clone library were detected. In conclusion, this prototype 16S rRNA-based taxonomic microarray appears to be a promising tool for the analysis of Alphaproteobacteria in complex ecosystems.     

Potential of a 16S rRNA-based taxonomic microarray for analyzing the rhizosphere effects of maize on Agrobacterium spp. and bacterial communities

Bacterial diversity is central to ecosystem sustainability and soil biological function, for which the role of roots is important. The high-throughput analysis potential of taxonomic microarray should match the breadth of bacterial diversity. Here, the power of this technology was evidenced through methodological verifications and analysis of maize rhizosphere effect based on a 16S rRNA-based microarray developed from the prototype of H. Sanguin et al. (Environ. Microbiol. 8:289-307, 2006). The current probe set was composed of 170 probes (41 new probes in this work) that targeted essentially the Proteobacteria. Cloning and sequencing of 16S rRNA amplicons were carried out on maize rhizosphere and bulk soil DNA. All tested clones that had a perfect match with corresponding probes were positive in the hybridization experiment. The hierarchically nested probes were reliable, but the level of taxonomic identification was variable, depending on the probe set specificity. The comparison of experimental and theoretical hybridizations revealed 0.91% false positives and 0.81% false negatives. The microarray detection threshold was estimated at 0.03% of a given DNA type based on DNA spiking experiments. A comparison of the maize rhizosphere and bulk soil hybridization results showed a significant rhizosphere effect, with a higher predominance of Agrobacterium spp. in the rhizosphere, as well as a lower prevalence of Acidobacteria, Bacteroidetes, Verrucomicrobia, and Planctomycetes, a new taxon of interest in soil. In addition, well-known taxonomic groups such as Sphingomonas spp., Rhizobiaceae, and Actinobacteria were identified in both microbial habitats with strong hybridization signals. The taxonomic microarray developed in the present study was able to discriminate and characterize bacterial community composition in related biological samples, offering extensive possibilities for systematic exploration of bacterial diversity in ecosystems.     

Retrieval of nearly complete 16S rRNA gene sequences from environmental DNA following 16S rRNA-based community fingerprinting

16S rRNA-based fingerprinting techniques allow rapid analyses of overall bacterial community structure but suffer from a lack of phylogenetic information hitherto retrievable from the short 16S rRNA gene sequences obtained from excised bands. An approach is presented that allows nearly complete 16S rRNA gene sequences to be retrieved for abundant components of the bacterial community as obtained by the community fingerprint, i.e. those reflected by major fingerprint bands. This was achieved by designing a pair of highly specific primers derived from the sequence of an excised band. Combined with universal 16S rRNA primers, these specific primers were applied directly to environmental DNA serving as template. This procedure allowed the generation of a nearly complete 16S rRNA gene sequence of the target taxon by specific polymerase chain reaction (PCR) followed by cycle sequencing down to a relative abundance of at least 1.5% of the environmental DNA. The procedure was exemplified for an epsilonproteobacterium related to Thiomicrospira denitrificans occurring in the central Baltic Sea. This approach is based only on PCR without any cloning step involved. It allows focussing on specific target taxa and is thus rather efficient. This approach should be applicable in general to 16S rRNA or 16S rRNA gene-based fingerprinting techniques and their respective environmental DNA.     

Application and validation of DNA microarrays for the 16S rRNA-based analysis of marine bacterioplankton

An oligonucleotide probe-based DNA microarray was evaluated for its ability to detect 16S rRNA targets in marine bacterioplankton samples without prior amplification by polymerase chain reaction (PCR). The results obtained were compared with those of quantitative fluorescence in situ hybridization (FISH). For extraction and direct labelling of total RNA, a fast and efficient protocol based on commercially available kits was established. A set of redundant and hierarchically structured probes was applied, and specificity of hybridization was assessed by additional control oligonucleotides comprising single central mismatches. The protocol was initially tested by microarray analysis of bacterial pure cultures. Complete discrimination of all control oligonucleotides was achieved, indicating a high degree of hybridization specificity. In a co-culture, abundant members were detected by microarray analysis, but signal ratios of positive probes did not correlate well with quantitative data from FISH experiments. A marine picoplankton sample from the German Bight was analysed. Bacterial populations with relative abundances of at least 5% were detected by hybridizing 0.1 microg of total RNA extracted from a sample of 375 ml equivalent to 4.1 x 10(8) cells. Our results demonstrate that major populations of marine bacterioplankton can be identified by microarray analysis in a fast and reliable way, even in relatively low volumes of sea water.     

PCR amplification of 16S rDNA sequences in Fe-rich sediment of coal refuse drainage

Acid drainage from coal mines or coal refuse, when treated in collection ponds, results in the formation of Fe-rich sediments. The Fe in these sediments inhibits the PCR and hinders identification of bacteria using non-culture 16S rDNA molecular methods. We describe a technique that employs EDTA to dissolve and remove Fe from these sediments, yielding DNA that can be amplified by PCR. The technique allows the identification of bacterial DNA sequences and increases our understanding of the bacterial community in Fe-rich sediments.