International Nucleotide Sequence Database Research Articles

In memoriam –Scott Federhen (October 21, 1952 - May 5, 2016)

Scott (Herbert) Federhen passed away on May 5, 2016, after a short illness. Scott was well known for heading the National Center for Biotechnology Information’s (NCBI’s) Taxonomy project, which he started in 1991. Under his leadership it became an international standard for nomenclature and classification. Born in 1952, Scott obtained a MS degree in Computer Science, focused on denotational semantics of AI languages, from University of Maryland in 1980. An MS in Biology from MIT followed, with a focus on control structures in biological systems. He joined NCBI in 1990, shortly after its establishment. His dual training in both Computer Sciences and Biology served him well as he oversaw tremendous growth in the NCBI Taxonomy Database, developed numerous submission and database enhancements, and collaborated regularly with many other NCBI groups and members of the International Nucleotide Sequence Database Collaboration (INSDC). The INSDC continues to follow the NCBI Taxonomy Database classification. Under Scott’s guidance, the NCBI Taxonomy group was occasionally involved in scientific controversy, when underlying molecular data supported taxonomic changes. One example is the early inclusion of birds (Aves) under the Dinosauria in the NCBI classification. Scott also spearheaded NCBI’s efforts with the Barcode of Life project, an international initiative to identify and archive the barcode (a short genetic sequence from a standard part of the genome) for every species of life. Recently, Scott was instrumental in greatly expanding the annotation of type material in the NCBI taxonomic database. This allowed for improved taxonomic accuracy. Almost a year to the day before his death, he presented a proposal he developed for a groundbreaking change in how taxonomic names would be treated at NCBI. His sly sense of humor is evidenced in the title: “A Modest Proposal for making the Genomes of GenBank beneficial to the Publick, and preventing them from being a Burthen to the Curators and Taxonomists” (a nod to Johnathan Swift’s famous satire). The group he invited to the workshop included members of the International Committee on Systematics of Prokaryotes (ICSP) and the Judicial Commission on Prokaryotic Nomenclature, as well as bioinformaticians and taxonomists working with genomic similarity measures. Scott proposed a set of measures to represent a scaffold of reliably identified sequences that could be used to find and correct misidentified genomes. The invitees also agreed on a set of principles that will guide future efforts. It is anticipated that this project will continue and expand, but its inception was a testament to Scott’s drive and vision. Scott Federhen will be remembered as an adventurous eater, kind colleague and devoted Red Sox fan with an exceptionally broad intellectual range. He leaves behind his wife, Sheree, a daughter, Erica, a son, Luke, and many friends.

Reminder to deposit DNA sequences

As members of the Advisory Committee to the International Nucleotide Sequence Database Collaboration (INSDC), which includes the DNA Data Bank of Japan (DDBJ), ENA, and GenBank databases, we wish to remind the research community of the importance of depositing complete DNA-sequence data in these databases upon publication of their results [see also S. L. Salzberg et al., Nature , (2016)]. Indeed, most journals demand a database accession number as a condition of publication. Access to the INSDC's databases is free and unrestricted ([ 1 ][1]), enabling researchers to plan experiments and to analyze existing data. As original contributions, deposited data form part of the scientific record and are citable in the literature. Authors can also correct and update their data. These amended records may be removed from the next database release but still remain permanently available by accession number. INSDC has also created major new repositories for large data collections, notably the Sequence Read Archive at the National Center for Biotechnology Information (NCBI) ([ 2 ][2]), the DDBJ Sequence Read Archive ([ 3 ][3]), and the European Nucleotide Archive at the European Molecular Biology Laboratory's European Bioinformatics Institute (EMBL-EBI) ([ 4 ][4]). These archive raw data from sequencing experiments, a crucial facility for reproducibility and reuse. For papers dependent on sequence data from human subjects, unrestricted data release may not be possible. In these cases, we would encourage journal editors to insist on data sharing through other repositories that are not part of INSDC, such as NCBI's Database of Genotype and Phenotype ([ 5 ][5]), EMBL-EBI's European Genome-phenome Archive ([ 6 ][6]), or DDBJ's Japanese Genotype-phenotype Archive ([ 7 ][7]). ![Figure][8] IMAGE: BEHOLDINGEYE/[ISTOCKPHOTO.COM][9] 1. [↵][10]1. G. Cochrane, 2. I. Karsch-Mizrachi, 3. T. Takagi , International Nucleotide Sequence Database Collaboration, Nucleic Acids Res 44, D48 (2016). [OpenUrl][11] 2. [↵][12][www.ncbi.nlm.nih.gov/sra][13]. 3. [↵][14] . 4. [↵][15][www.ebi.ac.uk/ena][16]. 5. [↵][17][www.ncbi.nlm.nih.gov/gap][18]. 6. [↵][19][www.ebi.ac.uk/ega/home][20]. 7. [↵][21] . [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-4 [5]: #ref-5 [6]: #ref-6 [7]: #ref-7 [8]: pending:yes [9]: http://ISTOCKPHOTO.COM [10]: #xref-ref-1-1 View reference 1 in text [11]: {openurl}?query=rft.jtitle%253DInternational%2BNucleotide%2BSequence%2BDatabase%2BCollaboration%26rft.volume%253D44%26rft.spage%253DD48%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [12]: #xref-ref-2-1 View reference 2 in text [13]: http://www.ncbi.nlm.nih.gov/sra [14]: #xref-ref-3-1 View reference 3 in text [15]: #xref-ref-4-1 View reference 4 in text [16]: http://www.ebi.ac.uk/ena [17]: #xref-ref-5-1 View reference 5 in text [18]: http://www.ncbi.nlm.nih.gov/gap [19]: #xref-ref-6-1 View reference 6 in text [20]: http://www.ebi.ac.uk/ega/home [21]: #xref-ref-7-1 View reference 7 in text

Diversity and identification of fungi associated with needles of Pinus radiata in Tasmania

Needle fungi have been extensively studied in conifers but rarely in Pinus radiata. Previous studies of P. radiata have been based on fungal isolation and not direct PCR detection from needles. This research was a component of a study examining factors linked to spring needle cast (SNC) in Tasmanian P. radiata plantations and aimed to identify as many as possible of the fungal species commonly associated with P. radiata needles in Tasmanian plantations. Needle samples were collected from 13 sites representative of the range of sites in which P. radiata is grown in Tasmania. Fungi were detected by a direct PCR approach and identified using barcode sequences from a reference collection of isolates from Tasmania, mainland Australia and New Zealand as well as sequences from the international nucleotide sequence databases. The total number of molecular operational taxonomic units (MOTUs) was 152, with 127 detected by direct PCR and sequencing, and only 35 operational taxonomic units (OTUs) isolated by culture-based methods. Dothideomycetes was the most diverse class detected in this study, with many MOTUs detected by direct PCR and not isolated. Leotiomycetes was the second-most diverse class and Sordariomycetes third, with several OTUs frequently isolated but rarely or not at all detected by direct PCR. DNA sequence data facilitated the discrimination and identification of OTUs, but some effort was required to avoid confusion caused by poorly identified isolates in public DNA databases. This is the most comprehensive study yet of fungi associated with pine needles in Australia and highlights the hitherto unrecognised diversity of Teratosphaeriaceae in P. radiata.

NCBI-compliant genome submissions: tips and tricks to save time and money.

Genome sequences nowadays play a central role in molecular biology and bioinformatics. These sequences are shared with the scientific community through sequence databases. The sequence repositories of the International Nucleotide Sequence Database Collaboration (INSDC, comprising GenBank, ENA and DDBJ) are the largest in the world. Preparing an annotated sequence in such a way that it will be accepted by the database is challenging because many validation criteria apply. In our opinion, it is an undesirable situation that researchers who want to submit their sequence need either a lot of experience or help from partners to get the job done. To save valuable time and money, we list a number of recommendations for people who want to submit an annotated genome to a sequence database, as well as for tool developers, who could help to ease the process.

The International Nucleotide Sequence Database Collaboration

The International Nucleotide Sequence Database Collaboration (INSDC; http://www.insdc.org) comprises three global partners committed to capturing, preserving and providing comprehensive public-domain nucleotide sequence information. The INSDC establishes standards, formats and protocols for data and metadata to make it easier for individuals and organisations to submit their nucleotide data reliably to public archives. This work enables the continuous, global exchange of information about living things. Here we present an update of the INSDC in 2015, including data growth and diversification, new standards and requirements by publishers for authors to submit their data to the public archives. The INSDC serves as a model for data sharing in the life sciences.

Assembly: a resource for assembled genomes at NCBI.

The NCBI Assembly database (www.ncbi.nlm.nih.gov/assembly/) provides stable accessioning and data tracking for genome assembly data. The model underlying the database can accommodate a range of assembly structures, including sets of unordered contig or scaffold sequences, bacterial genomes consisting of a single complete chromosome, or complex structures such as a human genome with modeled allelic variation. The database provides an assembly accession and version to unambiguously identify the set of sequences that make up a particular version of an assembly, and tracks changes to updated genome assemblies. The Assembly database reports metadata such as assembly names, simple statistical reports of the assembly (number of contigs and scaffolds, contiguity metrics such as contig N50, total sequence length and total gap length) as well as the assembly update history. The Assembly database also tracks the relationship between an assembly submitted to the International Nucleotide Sequence Database Consortium (INSDC) and the assembly represented in the NCBI RefSeq project. Users can find assemblies of interest by querying the Assembly Resource directly or by browsing available assemblies for a particular organism. Links in the Assembly Resource allow users to easily download sequence and annotations for current versions of genome assemblies from the NCBI genomes FTP site.

DNA data bank of Japan (DDBJ) progress report.

The DNA Data Bank of Japan Center (DDBJ Center; http://www.ddbj.nig.ac.jp) maintains and provides public archival, retrieval and analytical services for biological information. The contents of the DDBJ databases are shared with the US National Center for Biotechnology Information (NCBI) and the European Bioinformatics Institute (EBI) within the framework of the International Nucleotide Sequence Database Collaboration (INSDC). Since 2013, the DDBJ Center has been operating the Japanese Genotype-phenotype Archive (JGA) in collaboration with the National Bioscience Database Center (NBDC) in Japan. In addition, the DDBJ Center develops semantic web technologies for data integration and sharing in collaboration with the Database Center for Life Science (DBCLS) in Japan. This paper briefly reports on the activities of the DDBJ Center over the past year including submissions to databases and improvements in our services for data retrieval, analysis, and integration.

Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation.

The RefSeq project at the National Center for Biotechnology Information (NCBI) maintains and curates a publicly available database of annotated genomic, transcript, and protein sequence records (http://www.ncbi.nlm.nih.gov/refseq/). The RefSeq project leverages the data submitted to the International Nucleotide Sequence Database Collaboration (INSDC) against a combination of computation, manual curation, and collaboration to produce a standard set of stable, non-redundant reference sequences. The RefSeq project augments these reference sequences with current knowledge including publications, functional features and informative nomenclature. The database currently represents sequences from more than 55 000 organisms (>4800 viruses, >40 000 prokaryotes and >10 000 eukaryotes; RefSeq release 71), ranging from a single record to complete genomes. This paper summarizes the current status of the viral, prokaryotic, and eukaryotic branches of the RefSeq project, reports on improvements to data access and details efforts to further expand the taxonomic representation of the collection. We also highlight diverse functional curation initiatives that support multiple uses of RefSeq data including taxonomic validation, genome annotation, comparative genomics, and clinical testing. We summarize our approach to utilizing available RNA-Seq and other data types in our manual curation process for vertebrate, plant, and other species, and describe a new direction for prokaryotic genomes and protein name management.

Current Knowledge on Hepatitis E.

Although only a single serotype of hepatitis E virus (HEV), the causative agent of hepatitis E, has been identified, there is great genetic variation among the different HEV isolates reported. There are at least four major recognized genotypes of HEV: genotypes 1 and 2 are mainly restricted to humans and linked to epidemic outbreaks in nonindustrialized countries, whereas genotypes 3 and 4 are zoonotic in both developing and industrialized countries. Besides human strains, genotype 3 and 4 strains of HEV have been genetically characterized from swine, sika deer, mongooses, sheep, and rabbits. Currently, there are approximately 11,000 human and animal sequences of HEV available at the International Nucleotide Sequence Database Collaboration. HEV is the major cause of waterborne outbreaks of hepatitis in areas of poor sanitation. Additionally, it is responsible for sporadic cases of viral hepatitis in not only endemic but industrialized countries as well. Transmission of HEV occurs predominantly by the fecal-oral route, although parenteral and perinatal routes have been reported. HEV infection develops in most individuals as a self-limiting, acute, icteric hepatitis; with mortality rates around 1%. However, some affected individuals will develop fulminant hepatic failure, a serious condition that is frequently fatal without a liver transplant. This complication is particularly common when the infection occurs in pregnant women, where mortality rates rise dramatically to up to 25%. Among the preventive measures available to avoid HEV infection, two separate subunit vaccines containing recombinant truncated capsid proteins of HEV have been shown to be highly effective in the prevention of disease. One of them, HEV 239, was approved in China, and its commercialization by Innovax began in November 2012 under the name Hecolin®.

Ignored sediment fungal populations in water supply reservoirs are revealed by quantitative PCR and 454 pyrosequencing.

BackgroundThe sediment hosts a variety of fungal species in water supply reservoirs; however, the taxonomically and functionally diverse fungal populations have remained vastly unexplored. Here, quantitative PCR (qPCR) and recently developed high-throughput 454 GS FLX pyrosequencing were combined to investigate the abundance and diversity of sediment fungal communities in three water supply reservoirs.ResultsThese results revealed 1991, 2473, and 2610 copies of the 18S rRNA gene in the sediments from the ZC, SBY, and JP reservoirs, respectively. The fungal abundance in JP reservoir was 1.31 times higher than that of the ZC reservoir. In general, 43123 reads were recovered, corresponding to 945 distinct molecular operational taxonomic units (OTUs, 97% similarity cut-off level). The majority of the fungal nuclear ribosomal internal transcribed spacer (ITS) region sequences were affiliated with Ascomycota, Chytridiomycota, Basidiomycota, Glomeromycota, and Mucoromycotina. The highest Chao 1 index (962) was observed in the JP reservoir, and this value was 5.66 times greater than that of the SBY reservoir. Heat map analysis showed that Rhizophydium (relative frequency 30.98%), Placidium (20.20%), Apophysomyces (8.43%), Allomyces (6.26%), and Rhodotorula (6.01%) were the dominant genera in the JP reservoir, while Elaphomyces (20.0%) was the dominant genus in the ZC reservoir and Rhizophydium (30.98%) and Mattirolomyces (39.40%) were the most abundant in the JP and SBY reservoirs. Glomus sp. was only found in the JP reservoir. Furthermore, the larger proportions of “unassigned fungi” call for crafting International Nucleotide Sequence Database. Principle component analysis (PCA) and network analysis also suggested that tremendously diverse functional fungal populations were resident in the sediments of the three water supply reservoirs.ConclusionsThus, the results of this research suggest that the combination of high-throughput Roche 454 GS FLX pyrosequencing and qPCR is successfully employed to decrypt reservoir sediment fungal communities. Diverse fungi occur widely in the sediments of water supply reservoirs. These findings will undoubtedly broaden our understanding of reservoir sediment fungal species harbored in this freshwater stressful environmental condition. Future research should be conducted to determine the potential for fungi to degrade complex pollutants and their secondary metabolites related to the water quality.

Towards the unification of sequence-based classification and sequence-based identification of host-associated microorganisms.

Towards the unification of sequence-based classification and sequence-based identification of host-associated microorganisms.

LncRNAdb v2.0: expanding the reference database for functional long noncoding RNAs.

Despite the prevalence of long noncoding RNA (lncRNA) genes in eukaryotic genomes, only a small proportion have been examined for biological function. lncRNAdb, available at http://lncrnadb.org, provides users with a comprehensive, manually curated reference database of 287 eukaryotic lncRNAs that have been described independently in the scientific literature. In addition to capturing a great proportion of the recent literature describing functions for individual lncRNAs, lncRNAdb now offers an improved user interface enabling greater accessibility to sequence information, expression data and the literature. The new features in lncRNAdb include the integration of Illumina Body Atlas expression profiles, nucleotide sequence information, a BLAST search tool and easy export of content via direct download or a REST API. lncRNAdb is now endorsed by RNAcentral and is in compliance with the International Nucleotide Sequence Database Collaboration.

Improving ITS sequence data for identification of plant pathogenic fungi

Plant pathogenic fungi are a large and diverse assemblage of eukaryotes with substantial impacts on natural ecosystems and human endeavours. These taxa often have complex and poorly understood life cycles, lack observable, discriminatory morphological characters, and may not be amenable to in vitro culturing. As a result, species identification is frequently difficult. Molecular (DNA sequence) data have emerged as crucial information for the taxonomic identification of plant pathogenic fungi, with the nuclear ribosomal internal transcribed spacer (ITS) region being the most popular marker. However, international nucleotide sequence databases are accumulating numerous sequences of compromised or low-resolution taxonomic annotations and substandard technical quality, making their use in the molecular identification of plant pathogenic fungi problematic. Here we report on a concerted effort to identify high-quality reference sequences for various plant pathogenic fungi and to re-annotate incorrectly or insufficiently annotated public ITS sequences from these fungal lineages. A third objective was to enrich the sequences with geographical and ecological metadata. The results – a total of 31,954 changes – are incorporated in and made available through the UNITE database for molecular identification of fungi (http://unite.ut.ee), including standalone FASTA files of sequence data for local BLAST searches, use in the next-generation sequencing analysis platforms QIIME and mothur, and related applications. The present initiative is just a beginning to cover the wide spectrum of plant pathogenic fungi, and we invite all researchers with pertinent expertise to join the annotation effort.

Toward richer metadata for microbial sequences: replacing strain-level NCBI taxonomy taxids with BioProject, BioSample and Assembly records.

Microbial genome sequence submissions to the International Nucleotide Sequence Database Collaboration (INSDC) have been annotated with organism names that include the strain identifier. Each of these strain-level names has been assigned a unique ‘taxid’ in the NCBI Taxonomy Database. With the significant growth in genome sequencing, it is not possible to continue with the curation of strain-level taxids. In January 2014, NCBI will cease assigning strain-level taxids. Instead, submitters are encouraged provide strain information and rich metadata with their submission to the sequence database, BioProject and BioSample.

RefSeq microbial genomes database: new representation and annotation strategy

The source of the microbial genomic sequences in the RefSeq collection is the set of primary sequence records submitted to the International Nucleotide Sequence Database public archives. These can be accessed through the Entrez search and retrieval system at http://www.ncbi.nlm.nih.gov/genome. Next-generation sequencing has enabled researchers to perform genomic sequencing at rates that were unimaginable in the past. Microbial genomes can now be sequenced in a matter of hours, which has led to a significant increase in the number of assembled genomes deposited in the public archives. This huge increase in DNA sequence data presents new challenges for the annotation, analysis and visualization bioinformatics tools. New strategies have been developed for the annotation and representation of reference genomes and sequence variations derived from population studies and clinical outbreaks.

DDBJ progress report: a new submission system for leading to a correct annotation

The DNA Data Bank of Japan (DDBJ; http://www.ddbj.nig.ac.jp) maintains and provides archival, retrieval and analytical resources for biological information. This database content is shared with the US National Center for Biotechnology Information (NCBI) and the European Bioinformatics Institute (EBI) within the framework of the International Nucleotide Sequence Database Collaboration (INSDC). DDBJ launched a new nucleotide sequence submission system for receiving traditional nucleotide sequence. We expect that the new submission system will be useful for many submitters to input accurate annotation and reduce the time needed for data input. In addition, DDBJ has started a new service, the Japanese Genotype–phenotype Archive (JGA), with our partner institute, the National Bioscience Database Center (NBDC). JGA permanently archives and shares all types of individual human genetic and phenotypic data. We also introduce improvements in the DDBJ services and databases made during the past year.

Towards a unified paradigm for sequence‐based identification of fungi

The nuclear ribosomal internal transcribed spacer (ITS) region is the formal fungal barcode and in most cases the marker of choice for the exploration of fungal diversity in environmental samples. Two problems are particularly acute in the pursuit of satisfactory taxonomic assignment of newly generated ITS sequences: (i) the lack of an inclusive, reliable public reference data set and (ii) the lack of means to refer to fungal species, for which no Latin name is available in a standardized stable way. Here, we report on progress in these regards through further development of the UNITE database (http://unite.ut.ee) for molecular identification of fungi. All fungal species represented by at least two ITS sequences in the international nucleotide sequence databases are now given a unique, stable name of the accession number type (e.g. Hymenoscyphus pseudoalbidus|GU586904|SH133781.05FU), and their taxonomic and ecological annotations were corrected as far as possible through a distributed, third-party annotation effort. We introduce the term 'species hypothesis' (SH) for the taxa discovered in clustering on different similarity thresholds (97-99%). An automatically or manually designated sequence is chosen to represent each such SH. These reference sequences are released (http://unite.ut.ee/repository.php) for use by the scientific community in, for example, local sequence similarity searches and in the QIIME pipeline. The system and the data will be updated automatically as the number of public fungal ITS sequences grows. We invite everybody in the position to improve the annotation or metadata associated with their particular fungal lineages of expertise to do so through the new Web-based sequence management system in UNITE.

Design and development of portal for biological database in agriculture

The application of novel and modern techniques in genetic engineering and genomics has resulted in information explosion in genomics. Three major genome databases under International Nucleotide Sequence Database collaboration NCBI, DDBJ and EMBL have been providing a convenient platform for submission of sequences which they share among themselves. Many institutes in India under Indian Council of Agricultural Research have scientists working on biotechnology and bioinformatics research. The various studies conducted by them, generate massive data related to biological information of plants, animals, insects, microbes and fisheries. These scientists are dependent on NCBI, EMBL, DDBJ and other portals for their sequence submissions, analysis and other data mining tasks. Due to various limitations imposed on these sites and the poor connectivity problem prevents them to conduct their studies on these open domain databases. The valued information generated by them needs to be shared by the scientific communities to eliminate the duplication of efforts and expedite their knowledge extended towards new findings. A secured common submission portal system with user-friendly interfaces, integrated help and error checking facilities has been developed in such a way that the database at the backend consists of a union of the items available on the above mentioned databases. Standard database management concepts have been employed for their systematic storage management. Extensive hardware resources in the form of high performance computing facility are being installed for deployment of this portal.Availabilityhttp://cabindb.iasri.res.in:8080/sequence_portal/

On the reliability of DNA sequences ofOphiocordyceps sinensisin public databases

Some DNA sequences in the International Nucleotide Sequence Databases (INSD) are erroneously annotated, which has lead to misleading conclusions in publications. Ophiocordyceps sinensis (syn. Cordyceps sinensis) is a fungus endemic to the Tibetan Plateau, and more than 100 populations covering almost its distribution area have been examined by us over recent years. In this study, using the data from authentic materials, we have evaluated the reliability of nucleotide sequences annotated as O. sinensis in the INSD. As of October 15, 2012, the INSD contained 874 records annotated as O. sinensis, including 555 records representing nuclear ribosomal DNA (63.5%), 197 representing protein-coding genes (22.5%), 92 representing random markers with unknown functions (10.5%), and 30 representing microsatellite loci (3.5%). Our analysis indicated that 39 of the 397 internal transcribed spacer entries, 27 of the 105 small subunit entries, and five of the 53 large subunit entries were incorrectly annotated as belonging to O. sinensis. For protein-coding sequences, all records of serine protease genes, the mating-type gene MAT1-2-1, the DNA lyase gene, the two largest subunits of RNA polymerase II, and elongation factor-1α gene were correct, while 14 of the 73 β-tubulin entries were indeterminate. Genetic diversity analyses using those sequences correctly identified as O. sinensis revealed significant genetic differentiation in the fungus although the extent of genetic differentiation varied with the gene. The relationship between O. sinensis and some other related fungal taxa is also discussed.

Evaluation of the authenticity of a highly novel environmental sequence from boreal forest soil using ribosomal RNA secondary structure modeling

The number of sequences from both formally described taxa and uncultured environmental DNA deposited in the International Nucleotide Sequence Databases has increased substantially over the last two decades. Although the majority of these sequences represent authentic gene copies, there is evidence of DNA artifacts in these databases as well. These include lab artifacts, such as PCR chimeras, and biological artifacts such as pseudogenes or other paralogous sequences. Sequences that fall in basal positions in phylogenetic trees and appear distant from known sequences are particularly suspect. Phylogenetic analyses suggest that a novel sequence type (NS1) found in two boreal forest soil clone libraries belongs to the fungal kingdom but does not fall unambiguously within any known phylum. We have evaluated this sequence type using an array of secondary-structure analyses. To our knowledge, such analyses have never been used on environmental ribosomal sequences. Ribosomal secondary structure was modeled for four rRNA loci (ITS1, 5.8S, ITS2, 5′ LSU). These models were analyzed for the presence of conserved domains, conserved nucleotide motifs, and compensatory base changes. Minimal free energy (MFE) foldings and GC contents of sequences representing the major fungal clades, as well as NS1, were also compared. NS1 displays secondary rRNA structures consistent with other fungi and many, but not all, conserved nucleotide motifs found across eukaryotes. However, our analyses show that many other authentic sequences from basal fungi lack more of these conserved motifs than does NS1. Together our findings suggest that NS1 represents an authentic gene copy. The methods described here can be used on any rRNA-coding sequence, not just environmental fungal sequences. As new-generation sequencing methods that yield shorter sequences become more widely implemented, methods that evaluate sequence authenticity should also be more widely implemented. For fungi, the adjacent 5.8S and ITS2 loci should be prioritized. This region is not only suited to distinguishing between closely related species, but it is also more informative in terms of expected secondary structure.