The primer is stored in sterile water. Search by lot number. Designed for sequencing inserts cloned into the M13 vectors and pUC plasmids. DNA cloned into this vector is constitutively expressed in mammalian cells; use the neomycin resistance gene for stable expression. We use these cookies to ensure our site functions securely and properly; they are necessary for our services to function and cannot be switched off in our systems.
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Find Sales Contact. Contact Us Customer Support. Add multiple products. Please Enquire This product is discontinued. Add to Helix This product is available through the Promega Helix onsite stocking program. The results of this study may be used as a guideline for selecting primer pairs with the best overall coverage and phylum spectrum for specific applications, therefore reducing the bias in PCR-based microbial diversity studies.
Understanding microbial diversity has been the ambition of scientists for decades. Because diversity analysis by cultivation is problematic for a significant fraction of Bacteria and Archaea , culture-independent surveys have been developed. With the advent of massive parallel sequencing technologies, direct sequencing of PCR amplicons became feasible 2—4. The continuous development of the technology, offering read lengths of up to bp nowadays, further improved throughput and resolution of 16S rDNA sequencing 7.
Since then, additional high-throughput sequencing technologies have become commercially available. The attractiveness of Illumina 8 lies in the reduced per base costs and comparatively high sequencing depth 9 , despite having short read lengths. While the major advantage of Ion Torrent 10 are its relatively low cost and rapid sequencing speed. For a detailed review of sequencing technologies please refer to Loman et al. There is no doubt that the rapid development of sequencing technologies has opened a new dimension in biodiversity analysis, but the diversity of technologies also adds complexity to the experimental design of a study.
The most critical step for accurate rDNA amplicon analysis is still the choice of primers 4 , Using suboptimal primers, or more precisely, primer pairs, can lead to under-representation 14 or selection against single species or even whole groups 15— Using inappropriate primers consequently leads to questionable biological conclusions 17— In this study, broad range 16S rDNA primers and primer pairs were investigated in silico with respect to overall coverage and phylum spectrum for Bacteria and Archaea as well as amplicon length.
For consistency, all primers were renamed according to the primer nomenclature suggested by Alm et al. Finally, the obtained results were compared with diversity estimates from previous metagenome studies Primers were renamed according to Alm et al.
Each name is composed of seven dash-separated parts, describing: the target gene, the rank of the target group, the target group, the target position within the gene, the primer version, the target strand and the length of the primer. An indication of the target gene. An indication of the largest taxonomic group targeted by the PCR primer.
An abbreviated description, limited to three to five letters, of the specific taxonomic or phylogenetic group targeted by the primer. A single lowercase letter indicating the version of the probe. A single uppercase letter indicating whether the probe sequence is identical to the DNA sense strand S or to the antisense A strand; and. A total of forward and reverse 16S rDNA primers were chosen for the in silico evaluation. All primers are available in probeBase, a comprehensive online database for rRNA-targeted oligonucleotides, at www.
Primer pairs were chosen according to annealing temperatures, overall coverage of variable regions and amplicon length. Annealing temperatures were calculated with OligoCalc Suitable primer pairs were organized into three different groups Supplementary Table S8 : Group Short Group S generates — bp fragments.
Group Middle Group M generates — bp fragments. A total of primer combinations were evaluated. Sequences are required to have a SINA 26 alignment quality value better than 50 Alignment was attempted with SINA for all GOS reads and all sequences with an alignment quality of at least 30 and a minimum length of were retained, yielding a dataset of 10 sequences.
Taxonomic classifications for each read were applied as described below. Primer matching was executed using the probe match function of the ARB PT server 30 at two levels of stringency, allowing zero or one mismatch, respectively.
For each primer and stringency level the database entries were separated into three groups: i matches; ii mismatches; and iii unknown. The match status was considered to be unknown if no sequence data was available at the match position of the respective primer.
Furthermore, only sequences corresponding to the primer at the intended position where considered to be matches.
From these numbers, coverage was computed as the matched fraction of entries either matches or mismatches, excluding entries for which the match status was unknown.
Individual coverages were computed for all taxa. When computing the combined coverage of forward and reverse primer pairs, an entry was considered to have unknown match status if the match status for either of the two primers was unknown.
Likewise, the pair was only considered to be a match if both primers matched at the intended match position. Detailed information for each analysed primer and primer pair are provided in the Supplementary Material Online single primer: Supplementary Tables S2 — S7 ; primer pairs: Supplementary Tables S9 — S All scripts and SQL queries as well as database dumps are available online at www.
Surface water was collected on 11 February and weekly from 31 March until October For harvesting a 0. At each time point 10 l and 15 l of seawater were filtered onto 8 filters for genomic DNA extraction. Details can be found in Teeling et al.
Results from 16S rDNA diversity analysis gained from metagenome studies of the same sampling dates 22 were used for comparison. Genomic DNA was directly extracted from filters as described in Zhou et al. In a minor modification to the protocol, no size selection of the fragments was performed. The fragments were subjected to end repair and polishing.
After subsequent emulsion PCR the fragment libraries were processed and sequenced according to the Roche protocols. The resulting sequences were processed using the standard Roche software for base calling, trimming of adaptors and quality trimming Genome Sequencer FLX System Software Manual version 2. Raw data were stored as FNA file. For metagenomics two full PTPs per sample were sequenced. Unaligned reads were not considered in downstream analysis to eliminate non 16S rDNA sequences.
Remaining PCR amplicons were separated based on the presence of aligned nucleotides at E. This strategy is robust against sequencing errors within the primer signatures or incomplete primer signatures. With this approach the need for barcoding during combined sequencing of 16S pyrotags derived from different PCR reactions on the same PTP lane was avoided.
FASTA files for each primer pair of the separated samples are available online at www. Reads of the filtered and separated 16S pyrotag datasets as well as metagenomes were dereplicated, clustered and classified on a sample by sample basis. Dereplication identification of identical reads ignoring overhangs was done with cd-hit-est of the cd-hit package 3.
Remaining sequences were clustered again with cd-hit-est using an identity criterion of 0. In the final step, the taxonomic path of each cluster reference read was mapped to the additional reads within the corresponding cluster plus the corresponding replicates as identified in the previous analysis step to finally obtain semi- quantitative information number of individual reads representing a taxonomic path.
Sequencing depth may infringe on the comparability of the resulting taxonomic resolution. To verify that the results derived from the 16S pyrotags were not an artefact of deep sequencing, the total number of 16S pyrotags was reduced until roughly equal amounts of classified pyrotags and classified metagenome reads remained for each sample.
Three subsets of each 16S pyrotag sample were adjusted by withdrawing equal amounts of sequences randomly without replacement. An analogue approach was described in Gilbert et al. The overall coverage of single primers was evaluated for all three domains of life Supplementary Table S1. Additionally for Bacteria and Archaea the phylum spectrum was investigated with respect to zero and one mismatch Supplementary Tables S2 — S5.
Eukaryota are only considered on domain level Supplementary Tables S5 — S6. At one-mismatch-stringency the total number increased to eligible primers. This is in line with a recent study by Wang and Qian The highest overall coverage and specificity for the domain Bacteria was detected for the primers S-D-Bacta-A A: 2.
Furthermore, 39 primers show relatively high overall coverage for more than one domain. Contrary to this, with only 6. Allowing one mismatch increases the overall coverage to A: A direct comparison of our results with the studies of Huws et al. Nossa et al. Walter et al. In respect to detailed phylum coverage Supplementary Tables S2 — S5 it should be noted that the numbers of sequences present in a phylum affects the values for phylum coverage. If the majority of a small phylum e. Caldiserica with 61 sequences is targeted, the coverage will be higher than for a member rich phylum e.
Firmicutes with 84 sequences. Similar effects occur for phyla in which only a small number of sequences contain sequence information at the primer position of interest. When combining forward and reverse primers, the bias of single primers can accumulate. To minimize the overall bias, primers with similar overall coverage and phylum spectrum must be used. In order to get suitable combinations for the different sequencing technologies, primer pairs were organized into three groups according to their amplicon length Supplementary Table S8.
Group S mall could be of particular interest for Illumina 8 and Ion Torrent 10 sequencing. Group L arge primer pairs are useful for sequencing methods such as PacBio 11 as well as for creating classical clone libraries. Again, the focus of this evaluation was Archaea and Bacteria. Eukaryota are only considered on domain level. Assuming that a standard PCR can tolerate up to two mismatches between the primer and its target 1 , results with one mismatch are also taken into account.
However, it should be noted that a primer mismatch can result in a biased picture of the bacterial diversity 41 and preferential amplification might lead to under-representation of important members of a community 14 , The best results with an overall coverage of This pair generates an amplicon length of bp which spans the hypervariable HV region three. With one mismatch allowed, overall coverage for Archaea increases to A: Nanoarchaeota and AAG show three mismatches. Hence, this primer pair shows the most promising results for Illumina and Ion Torrent sequencing.
If one mismatch is tolerated some Archaea A: These findings are in line with the conclusions of Baker and Cowan 42 , who claim that no domain-specific primer exists or can be designed that matches all bacterial 16S rDNA sequences.
This primer pair has a slightly lower overall coverage for Bacteria A: With one allowed mismatch A: Please note that one mismatch may also lead to amplification of archaeal 16S rDNA sequences. Hence, it is recommended for Bacteria. Within the bacterial domain, those two primer pairs cover 49 out of 59 phyla. The continuous failure of primers to detect Nanoachaeota is not surprising, due to the majority of Archaea - specific primers being designed prior to the discovery of the Nanoarchaeota Addition of Nanoarchaeota - specific primers 43 is recommended.
Note that these primers generate almost full-length sequences. In summary, both primer pairs can be recommended for amplification. They generate amplicons specific for Bacteria and Archaea with an average length of bp that spans the HV region four. No archaeal-specific primer pair achieves a full phylum spectrum Supplementary Table S This primer pair covers two out of eight phyla Crenarchaeota and Euryarchaeota , but the phylum spectrum increases remarkably to six detected phyla if one mismatch is allowed A: The frequent use of HV region six in diversity analysis makes this pair particularly interesting for comparative analysis 35 , 44 , For the domain Bacteria , several domain-specific primer pairs attain high overall coverage, but 27 out of 30 fail to detect more than 10 phyla Supplementary Table S Although the first two show higher overall coverage, the latter exhibits a larger phylum spectrum.
If one mismatch is tolerated A: However, some archaeal sequences are also detected. With no mismatches it only fails to detect Nanoachaeota and expands to full archaeal phylum spectrum if one mismatch is tolerated.
For Bacteria , an overall coverage of Allowing one mismatch results in an increased overall coverage A: In summary, with an amplicon length of bp and detection of HV regions 5—8 this primer pair qualifies to target Bacteria and Archaea.
This detailed evaluation also demonstrates that reverse and forward primers with individual high coverage do not automatically qualify as an optimal primer pair. Based on promising results within the human habitat, they suggested that this primer pair may be a good candidate to access the bacterial diversity in any habitat 1.
However, our evaluation reveals a lower overall coverage of A: 0. Even if one mismatch is allowed A: 0. The majority detects only the two sequence-rich phyla, Crenarchaeota and Euryarchaeota. Although performance increases slightly when one mismatch is allowed A: In summary, this primer pair cannot be recommended. Similar results are obtained for the other archaeal primer pairs of Group L.
The bacterial primer pairs show more satisfying results Supplementary Table S If one mismatch is allowed, overall coverage increases to A: 0. Remarkably, this is mostly compensated if one mismatch is allowed. Chlamydiae remains undetected due to three internal mismatches of the forward primer.
The promising results and excellent domain specificity of both primer pairs are depreciated by the fact that they only span HV regions 1—6 and 1—8, respectively. This domain-specific primer pair spans HV regions 1—9 and covers 52 out of 59 bacteria phyla.
One mismatch A: 0. Thus plenty of data for comparative analysis are available. Ideally, sequences obtained with a given primer should be excluded when evaluating that same primer.
In order to calibrate our previous analysis, re-evaluation of the results using the publicly available Global Ocean Sampling GOS database was performed. The initial GOS dataset consisted of 6. Although it is limited to the marine habitat, it is the most comprehensive dataset that provides a reasonable amount of relatively long fragments necessary for primer evaluation. However, the bacterial fraction was dominant, consisting of sequences, compared with only archaeal and eukaryotic 16S and 18S sequences, respectively.
Thus the results for Archaea and Eukaryota are uncertain and should only be seen as complementary information. In addition to the limited number of sequences, only a subset of phyla is present.
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