MiCA: A Web-Based Tool for the Analysis of Microbial Communities Based on Terminal-Restriction Fragment Length Polymorphisms of 16S and 18S rRNA Genes

A web-based resource, Microbial Community Analysis (MiCA), has been developed to facilitate studies on microbial community ecology that use analyses of terminal-restriction fragment length polymorphisms (T-RFLP) of 16S and 18S rRNA genes. MiCA provides an intuitive web interface to access two specialized programs and a specially formatted database of 16S ribosomal RNA sequences. The first program performs virtual polymerase chain reaction (PCR) amplification of rRNA genes and restriction of the amplicons using primer sequences and restriction enzymes chosen by the user. This program, in silico PCR and Restriction (ISPaR), uses a binary encoding of DNA sequences to rapidly scan large numbers of sequences in databases searching for primer annealing and restriction sites while permitting the user to specify the number of mismatches in primer sequences. ISPaR supports multiple digests with up to three enzymes. The number of base pairs between the 5′ and 3′ primers and the proximal restriction sites can be reported, printed, or exported in various formats. The second program, APLAUS, infers a plausible community structure(s) based on T-RFLP data supplied by a user. APLAUS estimates the relative abundances of populations and reports a listing of phylotypes that are consistent with the empirical data. MiCA is accessible at .     

引物应用策略：基于2对古菌16S rRNA基因通用引物对环境样品古菌多样性分析

【目的】找到适宜的16S rRNA基因通用引物应用策略,应对复杂环境微生物多样性调查,尤其目前高速发展的高通量测序技术带来的巨大挑战。【方法】用Oligocheck软件分别将两对应试的古菌16S rRNA基因通用引物与RDP(Ribosomal database project)数据库中古菌16S rRNA基因序列进行匹配比对。用两对应试引物分别构建海洋沉积物样品的古菌16S rRNA基因文库。【结果】软件匹配结果显示引物f109/r958与目的基因的匹配程度高于引物f21/r958。该结果与古菌16S rRNA基因文库RFLP分析、古菌多样性指数分析结果相吻合。数据还表明,2对引物的综合文库能更好满足该沉积物样品的古菌多样性分析。【结论】选用与数据库中目的基因匹配性高的通用引物和多个引物的联合使用,可以有效提高环境样品微生物多样性调查的分辨率。     

Validation of a universal set of primers to study animal-associated microeukaryotic communities

The application of metabarcoding to study animal-associated microeukaryotes has been restricted because the universal barcode used to study microeukaryotic ecology and distribution in the environment, the Small Subunit of the Ribosomal RNA gene (18S rRNA), is also present in the host. As a result, when host-associated microbial eukaryotes are analysed by metabarcoding, the reads tend to be dominated by host sequences. We have done an in silico validation against the SILVA 18S rRNA database of a non-metazoan primer set (primers that are biased against the metazoan 18S rRNA) that recovers only 2.6% of all the metazoan sequences, while recovering most of the other eukaryotes (80.4%). Among metazoans, the non-metazoan primers are predicted to amplify 74% of Porifera sequences, 4% of Ctenophora, and 15% of Cnidaria, while amplifying almost no sequences within Bilateria. In vivo, these non-metazoan primers reduce significantly the animal signal from coral and human samples, and when compared against universal primers provide at worst a 2-fold decrease in the number of metazoan reads and at best a 2800-fold decrease. This easy, inexpensive, and near-universal method for the study of animal-associated microeukaryotes diversity will contribute to a better understanding of the microbiome.     

Barcoding marine nematodes: an improved set of nematode 18S rRNA primers to overcome eukaryotic co-interference

Nematodes form an important component of many benthic marine ecosystems and DNA barcoding approaches could provide an insight into nematode community composition from different environments globally. We have amplified nematode 18S rRNA sequences using standard nematode18S rRNA primers from environmental DNA extracted from intertidal sediment collected from New Jersey coast, USA to test whether the published marine nematode 18S rRNA sequences from GenBank and EMBL databases can effectively assign unknown nematode sequences into genus or species level. Most of the sequenced clones showed some degree of identities with published marine nematode 18S rRNA sequences. However, relatively very few of the sequences could be assigned even to genus level based on sequence assignment rule. In addition, other eukaryotic 18S rRNA sequences were found to be co-amplified with commonly used nematode 18S rRNA primers. We found that the majority of the current nematode 18S rRNA primers will co-amplify other eukaryotes if environmental DNA is the target template. We therefore designed a new set of nematode 18S rRNA primers and evaluated them using environmental DNA in intertidal sediment from the New Jersey coast. In total, 40 clones were screened and subsequently sequenced and all the sequences showed varying degree of identities with published nematode 18S rRNA sequences from GenBank and EMBL databases, and no obvious eukaryotic co-amplicons were detected with new primers. Only 13 out of 40 clones amplified with the new primer set showed 100% identity to published Daptonema and Metachromadora 18S rRNA sequences. The current molecular databases for nematodes are dominated by sequences from NW Europe and need to be more extensively populated with new full length 18S rRNA nematode sequences collected from different biogeographic locations. The new primers developed in this study, in combination with an updated nematode 18S rRNA sequence database, would help us to better investigate and understand the diversity and community composition of free-living marine nematodes based on DNA barcoding approaches during biodiversity or biomonitoring surveys on a global-scale.     

The activity of bacteriocins from Carnobacterium maltaromaticum UAL307 against gram-negative bacteria in combination with EDTA treatment

Molecular microbial ecology studies are heavily reliant on 'Universal' 16S rRNA gene primers for elucidating microbial community structure and composition, and yet primer design and optimization is often overlooked. Primers that exhibit minor biases due to primer-template mismatches can substantially alter the pool of amplicons from a community DNA sample, resulting in inaccurate conclusions. As a result, it is important that primers are critically evaluated against the most comprehensive data sets available before commencing molecular microbial community studies. We present a user-friendly, multi-platform (e.g. Windows, Linux, Mac) method named spyder for the in silico design and assessment of 16S rRNA gene primers. The method utilizes the Ribosomal Database Project's Probe Match feature coupled with a compact program (available at http://people.uleth.ca/~selibl/Spyder/Spyder.html) that aligns and identifies mismatches between primers and templates. To demonstrate the value of spyder, we assessed commonly used 'Universal' and phyla-specific primers and identified primer modifications that improved the coverage of target organisms by 5-42% as well as removed excessive degeneracies.     

16S/18S/ITS扩增子高通量测序引物的生物信息学评估和改进

【背景】在过去的十几年里,基于核糖体RNA基因的扩增子测序技术被广泛用于各种生态系统中微生物群落的多样性检测。扩增子测序的使用极大地促进了土壤、水体、空气等环境中微生物生态的相关研究。【目的】随着高通量测序技术的不断发展和参考数据库的不断更新,针对不同的环境样本的引物选择和改进仍然需要更深入的校验。【方法】本文收集了目前在微生物群落研究中被广泛采用的标记基因扩增通用引物,包括16S rRNA基因扩增常用的8对通用引物和2对古菌引物、9对真菌转录间隔区(internal transcribed spacer,ITS)基因扩增引物,以及18S rRNA基因扩增的4对真核微生物通用引物和1对真菌特异性引物。这些引物中包括了地球微生物组计划(Earth Microbiome Project,EMP)推荐的2对16S rRNA基因扩增引物、1对ITS1基因扩增引物和1对18S rRNA基因扩增引物。采用最近更新的标准数据库对这些引物进行了覆盖度和特异性评价。【结果】EMP推荐的引物依然具有较高的覆盖度,而其他引物在覆盖度及对特定环境或类群的特异性上也各有特点。此外,最近有研究对这些通用引物进行了一些改进,而我们也发现,一个碱基的变化都可能会导致评价结果或扩增产物发生明显变化,简并碱基的引入既可以覆盖更多的物种,但同时也会在一定程度上降低关注物种的特异性。【结论】研究结果为微生态研究中标记基因的引物选择提供了一个广泛的指导,但在关注具体科学问题时,引物的选择仍需数据指导与实验尝试。     

Group‐specific polymerase chain reaction amplification of SSU rRNA‐encoding gene fragments from 12 microbial taxa

Starting from an alignment of all known representatives in GenBank, we designed group specific primers targeting SSU rRNA‐encoding sequences of 12 microbial taxa known to contain insect pathogens and symbionts. We tested the specificity of these primers using representative species of all 12 groups as control templates. Polymerase chain reaction amplification conditions were modified until only group‐specific templates yielded a positive signal. The presented primer pairs thus allow for the amplification of SSU rRNA‐encoding sequences representing specific microbial groups directly from the environment (a social insect host in our study). We discuss possible applications of the identified molecular tools.     

PrimerHunter: a primer design tool for PCR-based virus subtype identification

Rapid and reliable virus subtype identification is critical for accurate diagnosis of human infections, effective response to epidemic outbreaks and global-scale surveillance of highly pathogenic viral subtypes such as avian influenza H5N1. The polymerase chain reaction (PCR) has become the method of choice for virus subtype identification. However, designing subtype-specific PCR primer pairs is a very challenging task: on one hand, selected primer pairs must result in robust amplification in the presence of a significant degree of sequence heterogeneity within subtypes, on the other, they must discriminate between the subtype of interest and closely related subtypes. In this article, we present a new tool, called PrimerHunter, that can be used to select highly sensitive and specific primers for virus subtyping. Our tool takes as input sets of both target and nontarget sequences. Primers are selected such that they efficiently amplify any one of the target sequences, and none of the nontarget sequences. PrimerHunter ensures the desired amplification properties by using accurate estimates of melting temperature with mismatches, computed based on the nearest neighbor model via an efficient fractional programming algorithm. Validation experiments with three avian influenza HA subtypes confirm that primers selected by PrimerHunter have high sensitivity and specificity for target sequences.     

Assessment of fungal diversity in deep-sea sediments by multiple primer approach

Increasing evidence of the fungal diversity in deep-sea sediments has come from amplification of environmental DNA with fungal specific or eukaryote primer sets. In order to assess the fungal diversity in deep-sea sediments of the Central Indian Basin (CIB) at ~5,000 m depth, we amplified sediment DNA with four different primer sets. These were fungal-specific primer pair ITS1F/ITS4 (internal transcribed spacers), universal 18S rDNA primers NS1/NS2, Euk18S-42F/Euk18S-1492R and Euk18S-555F/Euk18S-1269R. One environmental library was constructed with each of the primer pairs, and 48 clones were sequenced per library. These sequences resulted in 8 fungal Operational Taxonomic Units (OTUs) with ITS and 19 OTUs with 18S rDNA primer sets respectively by taking into account the 2% sequence divergence cut-off for species delineation. These OTUs belonged to 20 distinct fungal genera of the phyla Ascomycota and Basidiomycota. Seven sequences were found to be divergent by 79–97% from the known sequences of the existing database and may be novel. A majority of the sequences clustered with known sequences of the existing taxa. The phylogenetic affiliation of a few fungal sequences with known environmental sequences from marine and hypersaline habitat suggests their autochthonous nature or adaptation to marine habitat. The amplification of sequences belonging to Exobasidiomycetes and Cystobasidiomycetes from deep-sea is being reported for the first time in this study. Amplification of fungal sequences with eukaryotic as well as fungal specific primers indicates that among eukaryotes, fungi appear to be a dominant group in the sampling site of the CIB.     

Novel primers for 16S rRNA-based archaeal community analyses in environmental samples

Next generation sequencing technologies for in depth analyses of complex microbial communities rely on rational primer design based on up-to-date reference databases. Most of the 16S rRNA-gene based analyses of environmental Archaea community composition use PCR primers developed from small data sets several years ago, making an update long overdue. Here we present a new set of archaeal primers targeting the 16S rRNA gene designed from 8500 aligned archaeal sequences in the SILVA database. The primers 340F-1000R showed a high archaeal specificity (< 1% bacteria amplification) covering 93 and 97% of available sequences for Crenarchaeota and Euryarchaeota respectively. In silico tests of the primers revealed at least 38% higher coverage for Archaea compared to other commonly used primers. Empirical tests with clone libraries confirmed the high specificity of the primer pair to Archaea in three biomes: surface waters in the Arctic Ocean, the pelagic zone of a temperate lake and a methanogenic bioreactor. The clone libraries featured both Euryarchaeota and Crenarchaeota in variable proportions and revealed dramatic differences in the archaeal community composition and minimal phylogenetic overlap between samples.     

Group-specific PCR primers for the phylum Acidobacteria designed based on the comparative analysis of 16S rRNA gene sequences

We performed a comprehensive phylogenetic analysis of the phylum Acidobacteria and developed novel, group-specific PCR primers for Acidobacteria and its class-level subgroups. Acidobacterial 16S rRNA gene sequences deposited in the RDP database were used to construct a local database then subsequently analyzed. A total of 556 phylotypes were observed and the majority of the phylotypes belonged to five major subgroups (subgroups 1, 2, 3, 4, and 6), which comprised > 80% of the acidobacterial sequences in the RDP database. Phylum-specific and subgroup-specific primers were designed from the consensus sequences of the phylotype sequences, and the specificities of the designed primers were evaluated both in silico and empirically for coverage and tolerance. The phylum-specific primer ACIDO, which was designed in this study, showed increased coverage for Acidobacteria, as compared to the previous phylum-specific primer 31F. However, the tolerance of the primer ACIDO for non-target sequences was slightly higher than that of the primer 31F. We also developed subgroup-specific PCR primers for the major subgroups of Acidobacteria, except for subgroup 4. Subgroup-specific primers S1, S2, and S3, which targeted subgroups 1, 2, and 3, respectively, showed high coverage for their target subgroups and low tolerance for non-target sequences. However, the primer S6 targeting subgroup 6 showed a lower specificity in its empirical evaluation than expected from the in silico results. The subgroup-specific primers, as well as the phylum-specific primer designed in this study, will be valuable tools in understanding the phylogenetic diversity and ecological niche of the phylum Acidobacteria and its subgroups.     

Systematic Design of 18S rRNA Gene Primers for Determining Eukaryotic Diversity in Microbial Consortia

High-throughput sequencing of ribosomal RNA gene (rDNA) amplicons has opened up the door to large-scale comparative studies of microbial community structures. The short reads currently produced by massively parallel sequencing technologies make the choice of sequencing region crucial for accurate phylogenetic assignments. While for 16S rDNA, relevant regions have been well described, no truly systematic design of 18S rDNA primers aimed at resolving eukaryotic diversity has yet been reported. Here we used 31,862 18S rDNA sequences to design a set of broad-taxonomic range degenerate PCR primers. We simulated the phylogenetic information that each candidate primer pair would retrieve using paired- or single-end reads of various lengths, representing different sequencing technologies. Primer pairs targeting the V4 region performed best, allowing discrimination with paired-end reads as short as 150 bp (with 75% accuracy at genus level). The conditions for PCR amplification were optimised for one of these primer pairs and this was used to amplify 18S rDNA sequences from isolates as well as from a range of environmental samples which were then Illumina sequenced and analysed, revealing good concordance between expected and observed results. In summary, the reported primer sets will allow minimally biased assessment of eukaryotic diversity in different microbial ecosystems.     

Identifying group-specific primers for environmental Heterolobosa by high-throughput sequencing

Diversity of Heterolobosea (Excavata) in environments is poorly understood despite their ecological occurrence and health-associated risk, partly because this group tends to be under-covered by most universal eukaryotic primers used for sequencing. To overcome the limits of the traditional morpho-taxonomy-based biomonitoring, we constructed a primer database listing existing and newly designed specific primer pairs that have been evaluated for Heterolobosea 18S rRNA sequencing. In silico taxonomy performance against the current SILVA SSU database allowed the selection of primer pairs that were next evaluated on reference culture amoebal strains. Two primer pairs were retained for monitoring the diversity of Heterolobosea in freshwater environments, using high-throughput sequencing. Results showed that one of the newly designed primer pairs allowed species-level identification of most heterolobosean sequences. Such primer pair could enable informative, cultivation-free assays for characterizing heterolobosean populations in various environments.     

Potential bias of fungal 18S rDNA and internal transcribed spacer polymerase chain reaction primers for estimating fungal biodiversity in soil

Four fungal 18S rDNA and internal transcribed spacer (ITS) polymerase chain reaction (PCR) primer pairs were tested for their specificity towards target fungal DNA in soil DNA extracts, and their ability to assess the diversity of fungal communities in a natural grassland soil was compared. Amplified PCR products were cloned, and approximately 50 clones from each library were sequenced. Phylogenetic analysis and database searches indicated that each of the sequenced cloned DNA fragments was of fungal origin for each primer pair, with the exception of the sequences generated using the 18S rDNA primers nu-SSU-0817 and nu-SSU-1196, where 35 of the 50 sequenced clones represented soil invertebrates. Although some of the primers have previously been suggested to be biased towards certain fungal taxonomic groups, the ratio of sequences representing each of the four main fungal phyla, Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota, was similar for each of the primer pairs, suggesting that primer bias may be less significant than previously thought. Collector's curves were plotted to estimate the coverage obtained for each of the clone libraries after clustering the sequences into operational taxonomic units at a level of 99% sequence similarity. The curves indicated that good coverage of diversity was achieved, with the exception of the clone library constructed using primers nu-SSU-0817 and nu-SSU-1196, on account of the high number of non-fungal sequences obtained. The work demonstrates the usefulness of 18S rDNA and ITS PCR primers for assessing fungal diversity in environmental samples, and it also highlights some potential limitations of the approach with respect to PCR primer specificity and bias.     

Coamplification of Eukaryotic DNA with 16S rRNA Gene-Based PCR Primers: Possible Consequences for Population Fingerprinting of Complex Microbial Communities

The main aim of this study was to evaluate the specificity of three commonly used 16S rRNA gene-based polymerase chain reaction (PCR) primer sets for bacterial community analysis of samples contaminated with eukaryotic DNA. The specificity of primer sets targeting the V₃, V₃-V₅, and V₆-V₈ hypervariable regions of the 16S rRNA gene was investigated in silico and by community fingerprinting of human and fish intestinal samples. Both in silico and PCR-based analysis revealed cross-reactivity of the V₃ and V₃-V₅ primers with the 18S rRNA gene of human and sturgeon. The consequences of this primer anomaly were illustrated by denaturing gradient gel electrophoresis (DGGE) profiling of microbial communities in human feces and mixed gut of Siberian sturgeon. DGGE profiling indicated that the cross-reactivity of 16S rRNA gene primers with nontarget eukaryotic DNA might lead to an overestimation of bacterial biodiversity. This study has confirmed previous sporadic indications in literature indicating that several commonly applied 16S rRNA gene primer sets lack specificity toward bacteria in the presence of eukaryotic DNA. The phenomenon of cross-reactivity is a potential source of systematic error in all biodiversity studies where no subsequent analysis of individual community amplicons by cloning and sequencing is performed.     

Complementary retrieval of 16S rRNA gene sequences using broad-range primers with inosine at the 3'-terminus: implications for the study of microbial diversity

We evaluated the impact of the base analogue inosine substituted at the 3'-terminus of broad-range 16S rRNA gene primers on the recovery of microbial diversity using terminal restriction fragment length polymorphism and clonal analysis. Oral plaque biofilms from 10 individuals were tested with modified and unmodified primer pairs. Besides a core overlap of shared terminal restriction fragments (T-RFs), each primer system provided unique information on the occurrence of T-RFs, with a higher number generally displayed with inosine primers. All clones sequenced were at least 99% identical to publicly available full-length sequences. Analysis of the corresponding primer-binding sites showed that most sequence types were 100% complementary to the unmodified primers so that the characteristic of inosine to bind with all four nucleotides was not crucial for the observed increase in microbial richness. Instead, differences in community compositions were correlated with the identity of the nearest-neighbor 3' of the primer-targeting region. By influencing the thermal stability of primer hybridization, this position may play a previously unrecognized role in biased amplification of 16S rRNA gene sequences. In conclusion, the combined use of inosine and unmodified primers enables the complementary retrieval of 16S rRNA gene types, thereby expanding the observed diversity of complex microbial communities.     

Evaluation of PCR Primer Selectivity and Phylogenetic Specificity by Using Amplification of 16S rRNA Genes from Betaproteobacterial Ammonia-Oxidizing Bacteria in Environmental Samples

The effect of primer specificity for studying the diversity of ammonia-oxidizing betaproteobacteria (βAOB) was evaluated. βAOB represent a group of phylogenetically related organisms for which the 16S rRNA gene approach is especially suitable. We used experimental comparisons of primer performance with water samples, together with an in silico analysis of published sequences and a literature review of clone libraries made with four specific PCR primers for the βAOB 16S rRNA gene. With four aquatic samples, the primers NitA/NitB produced the highest frequency of ammonia-oxidizing-bacterium-like sequences compared to clone libraries with products amplified with the primer combinations βAMOf/βAMOr, βAMOf/Nso1255g, and NitA/Nso1225g. Both the experimental examination of ammonia-oxidizing-bacterium-specific 16S rRNA gene primers and the literature search showed that neither specificity nor sensitivity of primer combinations can be evaluated reliably only by sequence comparison. Apparently, the combination of sequence comparison and experimental data is the best approach to detect possible biases of PCR primers. Although this study focused on βAOB, the results presented here more generally exemplify the importance of primer selection and potential primer bias when analyzing microbial communities in environmental samples.     

Single nucleotide polymorphism-mismatch primer development for rapid molecular identification of selected microalgal species

Microalgae have been a great source for food, cosmetic, pharmacological, and biofuel production. The adoption of effective diagnostic assays for monitoring all stages of algal cultivation has become essential. In addition to microscopy identification, molecular assays can aid greatly in the identification and monitoring of algal species of interest. In this study the 18S ribosomal RNA (rRNA) sequences of 12 microalgal species and/or strains were used to design algal identification primers. Sequence alignment revealed five highly variable regions and multiple unique single nucleotide polymorphisms (SNPs). To design target algae specific primers, a SNP identified as unique to each microalgal species was incorporated into the 3’-terminus of forward and reverse primer pairs, respectively. To further enhance primer specificity, transverse mutation was introduced into each primer at the third base upstream of the respective SNP. The SNP-mismatch primer pairs yield size-specific amplicons, enabling the rapid molecular detection of 12 microalgae by circumventing cloning and sequencing. To verify the primer specificity, two SNP-mismatch primer pairs designed for Chlorella sorokiniana DOE1412 and wildtype species of Scenedesmus were tested in the outdoor reactor run inoculated with C. sorokiniana DOE1412. The primer pairs were able to identify C. sorokiniana DOE1412 as well as the environmental invader Scenedesmus sp. Furthermore, the “relative concentration” of two microalgae was accessed throughout the entire cultivation run. The use of SNPs primers designed in this study offers a cost-effective, easy to use alternative for routine monitoring of microalgal cultures in laboratories, in scale-ups, and in cultivation reactors, independent of the production platform.

     

Design and in vitro evaluation of new rpoB-DGGE primers for ruminants

Two new primer sets based on the rpoB gene were designed and evaluated with bovine and ovine rumen samples. The newly developed rpoB-DGGE primer set was used along with the 16S rRNA gene-V3, and another (old) rpoB-DGGE-based primer set from a previous study to in vitro compare the bovine and ovine rumen ecosystems. The results indicate a significant (P<0.001) difference in the microbial population between the two ruminants irrespective of the primers used in the analysis. Qualitative comparison of the data provides evidence for the presence of similar phyla profiles between the 16S rRNA gene and the newly developed rpoB primers. A comparison between the two rpoB-based primer sets (old and new) showed that the old rpoB-based primers failed to amplify phylum Bacteroidetes (a common phylum in the rumen) in both bovine and ovine rumen samples. The old and new rpoB-DGGE-based primers amplified a large number of clones belonging to phylum Proteobacteria, providing a useful insight into the microbial structure of the rumen. ChaoI, ACE, Simpson, and Shannon-Weaver index analysis estimated the bovine rumen to be more diverse than the ovine rumen for all three primer sets. These results provide a new insight into the community structure among ruminants using the newly developed primers in this study.     

Short pyrosequencing reads suffice for accurate microbial community analysis

Pyrosequencing technology allows us to characterize microbial communities using 16S ribosomal RNA (rRNA) sequences orders of magnitude faster and more cheaply than has previously been possible. However, results from different studies using pyrosequencing and traditional sequencing are often difficult to compare, because amplicons covering different regions of the rRNA might yield different conclusions. We used sequences from over 200 globally dispersed environments to test whether studies that used similar primers clustered together mistakenly, without regard to environment. We then tested whether primer choice affects sequence-based community analyses using UniFrac, our recently-developed method for comparing microbial communities. We performed three tests of primer effects. We tested whether different simulated amplicons generated the same UniFrac clustering results as near-full-length sequences for three recent large-scale studies of microbial communities in the mouse and human gut, and the Guerrero Negro microbial mat. We then repeated this analysis for short sequences (100-, 150-, 200- and 250-base reads) resembling those produced by pyrosequencing. The results show that sequencing effort is best focused on gathering more short sequences rather than fewer longer ones, provided that the primers are chosen wisely, and that community comparison methods such as UniFrac are surprisingly robust to variation in the region sequenced.