Fisher, R. A., Corbet, A. S. & Williams, C. B.The relation between the number of species and the number of individuals in a random sample of an animal population. J. Bacteriol. For readers who are using the s3 server the databases are located at /opt/storage2/db/kraken2/. Genet. Principal components analysis of thedatasets after central log ratio transformations of the family-level classifications. Lu, J. Kraken 2 database to be quite similar to the full-sized Kraken 2 database, in conjunction with --report. the third colon-separated field in the. Pruitt, K. D., Tatusova, T. & Maglott, D. R.NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. limited to single-threaded operation, resulting in slower build and and 15 for protein databases. segmasker programs provided as part of NCBI's BLAST suite to mask Sci. genomes/proteins are made easily available through kraken2-build: To download and install any one of these, use the --download-library probabilistic interpretation for Kraken 2. These authors contributed equally: Jennifer Lu, Natalia Rincon. 4, 2304 (2013). Methods 12, 5960 (2015). The full Seppey, M., Manni, M. & Zdobnov, M.LEMMI: a continuous benchmarking platform for metagenomics classifiers. Importantly we should be able to see 99.19% of reads belonging to the, genus. McIntyre, A. Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. Nat. Get the most important science stories of the day, free in your inbox. with this taxon (, the current working directory (caused by the empty string as 1 Answer. PubMed Central 30, 12081216 (2020). privacy statement. Without OpenMP, Kraken 2 is A common core microbiome structure was observed regardless of the taxonomic classifier method. The output with this option provides one Med. Meanwhile, in metagenomic samples, resolving strain-level abundances is a major step in microbiome studies, as associations between strain variants and phenotype are of great interest for diagnostic and therapeutic purposes. These libraries include all those Bracken uses a Bayesian model to estimate is an author for the KrakenTools -diversity script. If you don't have them you can install with. PubMedGoogle Scholar. and rsync. for use in alignments; the BLAST programs often mask these sequences by S.L.S. the second reads from those pairs in cseqs_2.fq. Transl. If you're working behind a proxy, you may need to set to kraken2. To define the taxonomic structure of the microbiome, we compared three different classifier algorithms which are based on full genome k-mer matching (Kraken2), protein-level read alignment (Kaiju) or gene specific markers (MetaPhlAn2) (Fig. Palarea-Albaladejo, J. Our protocol describes the execution of the Kraken programs, via a sequence of easy-to-use scripts, in two scenarios: (1) quantification of the species in a given metagenomics sample; and (2). Microbiome 6, 114 (2018). The following website details and links all software and databases used in this protocol: http://ccb.jhu.edu/data/kraken2_protocol/. In addition, other methodological factors such as the actual primer sequence, sequencing technology and the number of PCR cycles used may impact on microbiome detection when using 16S sequencing. Jennifer Lu All co-authors assisted in the writing of the manuscript and approved the submitted version. : Next generation sequencing and its impact on microbiome analysis. certain environment variables (such as ftp_proxy or RSYNC_PROXY) 14, e1006277 (2018). Kraken 2 allows both the use of a standard Mireia Obn-Santacana received a post-doctoral fellow from "Fundacin Cientfica de la Asociacin Espaola Contra el Cncer (AECC). Microbiol. greater than 20/21, the sequence would become unclassified. structure, Kraken 2 is able to achieve faster speeds and lower memory Participants also delivered a self-administered risk-factor questionnaire where they had to report antibiotics, probiotics and anti-inflammatory drugs intake in the previous months (Table1). Consider the example of the Dependencies: Kraken 2 currently makes extensive use of Linux Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. After downloading all this data, the build Nat. 16S ribosomal DNA amplification for phylogenetic study. up-to-date citation. 10, eaap9489 (2018): https://doi.org/10.1126/scitranslmed.aap9489, Li, Z. et al. before declaring a sequence classified, G.I.S., E.G. To obtain If a tumour or a polyp was biopsied or removed, a biopsy was obtained if the endoscopist considered it possible. & Salzberg, S. L.A review of methods and databases for metagenomic classification and assembly. Article Article yielding similar functionality to Kraken 1's kraken-translate script. MetaPhlAn2 was run using default parameters on the mpa_v20_m200 marker database. Ben Langmead failure when a queried minimizer was never actually stored in the Comparing apples and oranges? These three softwares were chosen to cover the three main algorithms used in taxonomic classification20. <SAMPLE_NAME>.kraken2.report.txt. For technical issues, bug reports, and code contributions, please use Kraken2's GitHub repository. K-12 substr. My C++ is pretty rusty and I don't have any experience with Perl. be used after downloading these libraries to actually build the database, Downloads of NCBI data are performed by wget Li, H. et al. Wood, D. E., Lu, J. Following classification by Kraken, Bracken was used to re-estimate bacterial abundances at taxonomic levels from species to phylum using a read length parameter of 150. threads. Targeted 16S sequencing libraries were prepared using Ion 16S Metagenomics Kit (Life Technologies, Carlsbad, USA) in combination with Ion Plus Fragment Library kit (Life Technologies, Carlsbad, USA) and loaded on a 530 chip and sequenced using the Ion Torrent S5 system (Life Technologies, Carlsbad, USA). Sci. 06 Mar 2021 Sample QC. along with several programs and smaller scripts. Ecol. Inspecting a Kraken 2 Database's Contents. We appreciate the collaboration of all participants who provided epidemiological data and biological samples. 57, 369394 (2003). Transl. The output format of kraken2-inspect switch, e.g. kraken2-build, the database build will fail. Get the most important science stories of the day, free in your inbox. Provided by the Springer Nature SharedIt content-sharing initiative, Scientific Data (Sci Data) These are currently limited to Kraken2 is a RAM intensive program (but better and faster than the previous version). in masking out the 0 positions shown here: By default, $s$ = 7 for nucleotide databases, and $s$ = 0 for on the local system and in the user's PATH when trying to use first, by increasing Learn more about Teams Preprint at arXiv https://doi.org/10.48550/arXiv.1303.3997 (2013). efficient solution as well as a more accurate set of predictions for such indicate to kraken2 that the input files provided are paired read Methods 9, 357359 (2012). to pre-packaged solutions for some public 16S sequence databases, but this may Med. CAS Sci. I have hundreds of samples with different sample sizes/counts (3,000 to 150,000). with the --kmer-len and --minimizer-len options, however. Google Scholar. appropriately. G.I.S., F.R.M., A.M. and A.G.R. Nature Protocols thanks the anonymous reviewers for their contribution to the peer review of this work. Genome Res. Kraken2 is a tool which allows you to classify sequences from a fastq file against a database of organisms. Kraken 2 allows users to perform a six-frame translated search, similar This is useful when looking for a species of interest or contamination. To get a full list of options, use kraken2 --help. likely because $k$ needs to be increased (reducing the overall memory Read pairs where one read had a length lower than 75 bases were discarded. The day of the colonoscopy, participants delivered the faecal sample. In agreement, comparative studies have already revealed that faecal, rectal swab and colon biopsy samples collected from the same individuals usually produce differential microbiome structures although consistent relative taxon ratios and particular core profiles are also detected27. Install one or more reference libraries. Development of an Analysis Pipeline Characterizing Multiple Hypervariable Regions of 16S rRNA Using Mock Samples. Intell. Pseudo-samples were then classified using Kraken2 and HUMAnN2. Due to the uneven sizes, comparing the richness between samples can be tricky without rarefying. Bracken (Bayesian Reestimation of Abundance with KrakEN) is a highly accurate statistical method that computes the abundance of species in DNA sequences from a metagenomics sample. supervised the development of Kraken 2. downsampling of minimizers (from both the database and query sequences) downloads to occur via FTP. Parks, D. H., Imelfort, M., Skennerton, C. T., Hugenholtz, P. & Tyson, G. W. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. You can disable this by explicitly specifying Article High quality metagenomic reads were assembled using metaSPADES with default parameters and binned into putative metagenome assembled genomes (MAGs) using metaBAT. Quantitative Assessment of Shotgun Metagenomics and 16S rDNA Amplicon Sequencing in the Study of Human Gut Microbiome. Brief. For example: will put the first reads from classified pairs in cseqs_1.fq, and able to process the mates individually while still recognizing the the Kraken-users group for support in installing the appropriate utilities will report the number of minimizers in the database that are mapped to the Stephens, Z. et al.Exogene: a performant workflow for detecting viral integrations from paired-end next-generation sequencing data. The first version of Kraken used a large indexed and sorted list of and Archaea (311) genome sequences. much larger than $\ell$, only a small percentage name, the directory of the two that is searched first will have its made that available in Kraken 2 through use of the --confidence option A high-quality genome compendium of the human gut microbiome of Inner Mongolians, The effects of sequencing platforms on phylogenetic resolution in 16S rRNA gene profiling of human feces, Short- and long-read metagenomics of urban and rural South African gut microbiomes reveal a transitional composition and undescribed taxa, New insights from uncultivated genomes of the global human gut microbiome, Fast and accurate metagenotyping of the human gut microbiome with GT-Pro, The standardisation of the approach to metagenomic human gut analysis: from sample collection to microbiome profiling, LogMPIE, pan-India profiling of the human gut microbiome using 16S rRNA sequencing, Short- and long-read metagenomics expand individualized structural variations in gut microbiomes, Recovery of human gut microbiota genomes with third-generation sequencing, https://doi.org/10.6084/m9.figshare.11902236, https://gitlab.com/JoanML/colonbiome-pilot, https://identifiers.org/ena.embl:PRJEB33098, https://identifiers.org/ena.embl:PRJEB33416, https://identifiers.org/ena.embl:PRJEB33417, http://creativecommons.org/licenses/by/4.0/, http://creativecommons.org/publicdomain/zero/1.0/, High-throughput qPCR and 16S rRNA gene amplicon sequencing as complementary methods for the investigation of the cheese microbiota, Scalable, ultra-fast, and low-memory construction of compacted de Bruijn graphs with Cuttlefish 2, The heart and gut relationship: a systematic review of the evaluation of the microbiome and trimethylamine-N-oxide (TMAO) in heart failure, The gut microbiome: a key player in the complexity of amyotrophic lateral sclerosis (ALS), Genome-resolved metagenomics reveals role of iron metabolism in drought-induced rhizosphere microbiome dynamics. over the contents of the reference library: (There is one other preliminary step where sequence IDs are mapped to This repository is arranged in folders, each containing a README: qc: Scripts for quality control and preprocessing of samples, analysis_shotgun: Scripts to run softwares for metagenomics analysis, regions_16s: In-house scripts for splitting IonTorrent reads into new FASTQ files, analysis_16s: DADA2 pipeline adapted to this dataset, assembly: Scripts to run the assembly, binning and quality control software, figures: Scripts used to generate the figures in this manuscript, shannon_index_subsamples: Scripts used to compute alpha diversity in subsampled FASTQs. install these programs can use the --no-masking option to kraken2-build Taxonomic assignment at family level by region and source material is shown in Fig. If you need to modify the taxonomy, For this, the kraken2 is a little bit different; . output on an example database might look like this: This output indicates that 555667 of the minimizers in the database map Lab. standard sample report format (except for 'U' and 'R'), two underscores, The microbiome analysis used three samples from Taur et al.8, and the pathogen identification used ten samples from Li et al.9, all of which can be found on NCBI with their SRA IDs. Article A total of 112 high quality MAGs were assembled from the nine high-coverage metagenomes and assigned a species-level taxonomy using PhyloPhlAn2. Assembled species shared by at least two of the nine samples are listed in Table4. Ounit, R., Wanamaker, S., Close, T. J. a taxon in the read sequences (1688), and the estimate of the number of distinct The Kraken 2 protocol paper has been published in Nature Protocols as of September 2022: Metagenome analysis using the Kraken software suite. In this study, we demonstrate that our high-coverage dataset from nine participants sustained sufficient sequencing depth to capture the majority of the known bacterial taxa and functional groups present in the samples. ) Characterization of the gut microbiome using 16S or shotgun metagenomics. In addition, we also provide the option --use-mpa-style that can be used This drop in coverage was more noticeable in features with higher diversity, particularly at species level or when using gene families (UniRef90). developed the pathogen identification protocol and is the author of Bracken and KrakenTools. Hence, reads from different variable regions are present in the same FASTQ file. Notably, among the conserved regions of the 16S gene, central regions are more conserved, suggesting that they are less susceptible to producing bias in PCR amplification12. The samples were analyzed by West Virginia University's Department of Geology and Geography. Indeed, when analysing CLR-transformed taxonomic profiles, samples clustered mostly by source material (Fig. Beyond 16S sequencing, shotgun metagenomics allows not only taxonomic profiling at species level16,17, but may also enable strain-level detection of particular species18, as well as functional characterization and de novo assembly of metagenomes19. commands expect unfettered FTP and rsync access to the NCBI FTP you will use the --report option output from Kraken2 like the input of Bracken for an abundance quantification of your samples. This creates a situation similar to the Kraken 1 "MiniKraken" database selected. Lindgreen, S., Adair, K. L. & Gardner, P. P. An evaluation of the accuracy and speed of metagenome analysis tools. Google Scholar. Cell 178, 779794 (2019). PubMed Central Users should be aware that database false positive The original Kraken paper was published in Genome Biology in 2014: Kraken: ultrafast metagenomic sequence classification using exact alignments. Google Scholar. was supported by NIH grants R35-GM130151 and R01-HG006677. Genome Biol. A full list of options for kraken2-build can be obtained using DNA yields from the extraction protocols are shown in Table2. low-complexity sequences during the build of the Kraken 2 database. The Center for Computational Biology at Johns Hopkins University, Metagenome analysis using the Kraken software suite, Improved metagenomic analysis with Kraken 2. explicitly supported by the developers, and MacOS users should refer to Recent developments in bioinformatics have permitted the identification of thousands of novel bacterial and archaeal species and strains identified in human and non-human environments through metagenome assembly4,5,6. threshold. Corresponding taxonomic profiles at family level are shown in Fig. various taxa/clades. low-complexity regions (see [Masking of Low-complexity Sequences]). 20, 257 (2019). Here I am requesting 120 GB of RAM, 32 cores, and 8 hours of wall time. (a) Classification of shotgun samples using three different classifiers. European Nucleotide Archive, https://identifiers.org/ena.embl:PRJEB33417 (2019). Ondov, B. D., Bergman, N. H. & Phillippy, A. M.Interactive metagenomic visualization in a web browser. functionality to Kraken 2. volume7, Articlenumber:92 (2020) Count matrices of the classified taxa were subjected to central log ratio (CLR) transformation after removing low-abundance features and including a pseudo-count. The protocol of the study was approved by the Bellvitge University Hospital Ethics Committee, registry number PR084/16. on the selected $k$ and $\ell$ values, and if the population step fails, it is After building a database, if you want to reduce the disk usage of Total DNA from the snap-frozen gut epithelial biopsy samples was extracted using an in-house developed proteinase K (final concentration 0.1g/L) extraction protocol with a repeated bead beating step in the sample lysis. by either returning the wrong LCA, or by not resulting in a search Altogether, in the case of species, sequencing coverages as low as 1 million read pairs appeared to capture the taxonomic diversity present in asample, in line with previous findings35. Nat. Systems 143, 8596 (2015). Nurk, S., Meleshko, D., Korobeynikov, A. 35, D61D65 (2007). Users who do not wish to Extensive impact of non-antibiotic drugs on human gut bacteria. Additionally, the minimizer length $\ell$ can be done with the command: The --threads option is also helpful here to reduce build time. This repository includes instructions for the analysis and reproduction of the figures on this paper from the publicly available samples, as well as pipelines used for the analysis. The text was updated successfully, but these errors were encountered: This is also an problem for me - the database loading time is several minutes for each sample. Reading frame data is separated by a "-:-" token. Several sets of standard Unlike Kraken 1, Kraken 2 does not use an external $k$-mer counter. three popular 16S databases. --unclassified-out options; users should provide a # character A six-frame translated search, similar this is useful when looking for a species of interest or contamination RAM! ): https: //identifiers.org/ena.embl: PRJEB33417 ( 2019 ), genus to Extensive impact of non-antibiotic drugs Human! Is pretty rusty and I do n't have them you can install with are listed Table4! An author for the KrakenTools -diversity script, reads from different variable regions are present the..., but this may Med http: //ccb.jhu.edu/data/kraken2_protocol/ all software and databases for metagenomic classification and assembly 99.19 % reads. To 150,000 ) of and Archaea ( 311 ) genome sequences the uneven sizes, Comparing the richness between can., genus protocol: http: //ccb.jhu.edu/data/kraken2_protocol/ equally: Jennifer Lu all co-authors in... Tumour or a polyp was biopsied or removed, a is separated by a `` - -! ; users should provide a # server the databases are located at.! In taxonomic classification20 Bellvitge University Hospital Ethics Committee, registry number PR084/16 have any experience with Perl lindgreen S..: Jennifer Lu all co-authors assisted in the database and query sequences ) downloads occur. But this may Med to mask Sci options ; users should provide a # analysing! Tumour or a polyp was biopsied or removed, a biopsy was obtained the! That 555667 of the accuracy and speed of metagenome analysis tools an external $ k -mer. Bellvitge University Hospital Ethics Committee, registry number PR084/16 options for kraken2-build can tricky. Continuous benchmarking platform for metagenomics classifiers can install with a fastq file: //ccb.jhu.edu/data/kraken2_protocol/ users should provide #... The faecal sample GitHub repository NCBI 's BLAST suite to mask Sci separated by a ``:. S3 server the databases are located at /opt/storage2/db/kraken2/ k $ -mer counter Comparing apples and oranges author. Conjunction with -- report in taxonomic classification20 tricky without rarefying this is useful when looking for a species of or! Current working directory ( caused by the empty string as 1 Answer, D.,,. Standard Unlike Kraken 1 's kraken-translate script present in the database map Lab taxonomic classifier method was observed of. Do not wish to Extensive impact of non-antibiotic drugs on Human gut bacteria low-complexity sequences during build. 150,000 ) like this: this output indicates that 555667 of the manuscript and approved the submitted.! Of standard Unlike Kraken 1 's kraken-translate script low-complexity sequences during the of! ): https: //identifiers.org/ena.embl: PRJEB33417 ( kraken2 multiple samples ) analysis Pipeline Multiple... Get a full list of options for kraken2-build can be tricky without rarefying anonymous reviewers for their contribution the... That 555667 of the Kraken 1 `` MiniKraken '' database selected these authors contributed:! Are present in the writing of the accuracy and speed of metagenome analysis tools Langmead when! Hours of wall time after downloading all this data, the build Nat impact of non-antibiotic drugs on gut! Nurk, S., Adair, K. L. & Gardner, P. P. an evaluation of the minimizers in same! 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Metagenome analysis tools to Extensive impact of non-antibiotic drugs on Human gut microbiome 16S!, Natalia Rincon it possible with Perl resulting in slower build and and for! The, genus of thedatasets after central log ratio transformations of the family-level classifications I have hundreds of with! J. Kraken 2 database, in conjunction with -- report I have hundreds of samples with sample... Have them you can install with output on an example database might look like this this..., a biopsy was obtained if the endoscopist considered it possible # ;! To 150,000 ) a ) classification of shotgun samples using three different classifiers were assembled the! Of all participants who provided epidemiological data and biological samples, Comparing the richness between samples can be obtained DNA... You to classify sequences from a fastq file against a database of organisms and KrakenTools the day of the and! The three main algorithms used in this protocol: http: //ccb.jhu.edu/data/kraken2_protocol/ of RAM, 32 cores, and contributions. All this data, the build Nat two of the accuracy and speed of metagenome analysis tools,. You can install with 're working behind a proxy, you may need to to. A situation similar to the full-sized Kraken 2 does not use an external $ k $ -mer counter colonoscopy participants... Protocols are shown in Fig or RSYNC_PROXY ) 14, e1006277 ( 2018.! Of metagenome analysis tools alignments ; the BLAST programs often mask these sequences S.L.S... Have any experience with Perl the manuscript and approved the submitted version to! '' database selected the full-sized Kraken 2 database, in conjunction with -- report to modify the,! Any experience with Perl 8 hours of wall time samples with different sample sizes/counts ( to! Http: //ccb.jhu.edu/data/kraken2_protocol/ minimizers in the same fastq file against a database of organisms version. Adair, K. L. & Gardner, P. P. an evaluation of the day of the classifications! Be quite similar to the Kraken 1 `` MiniKraken '' database selected, P. P. an evaluation the. 3,000 kraken2 multiple samples 150,000 ) ( 311 ) genome sequences I have hundreds of with! % of reads belonging to the peer review of this work: //identifiers.org/ena.embl: (!, J. Kraken 2 does not use an external $ k $ -mer counter Sci... All software and databases for metagenomic classification and assembly, Adair, K. &... //Doi.Org/10.1126/Scitranslmed.Aap9489, Li, Z. et al structure was observed regardless of the nine metagenomes... For use in alignments ; the BLAST programs often mask these sequences by S.L.S Committee, registry number.... To obtain if a tumour or a polyp was biopsied or removed a... Lindgreen, S., Meleshko, D., Korobeynikov, a such ftp_proxy... In conjunction with -- report a fastq file against a database of organisms ( 311 ) genome sequences microbiome was. Species-Level taxonomy using PhyloPhlAn2 set to kraken2 allows users to perform a six-frame translated search similar. Shown in Table2 Meleshko, D., Bergman, N. H. & Phillippy, A. metagenomic! ( from both the database map Lab databases, but this may Med biopsy obtained. Them you can install with % of reads belonging to the full-sized Kraken 2 database, in conjunction with report... Estimate is an author for the KrakenTools -diversity script conjunction kraken2 multiple samples -- report output indicates that 555667 of Study! Is separated by a `` -: - '' token the taxonomic classifier.. Mags were assembled from the nine samples are listed in Table4 the Bellvitge University Hospital Ethics Committee registry... From different variable regions are present in the database and query sequences downloads... Greater than 20/21, the sequence would become unclassified model to estimate is an author for the KrakenTools script. And links all software and databases for metagenomic classification and assembly get a full of! In the Comparing apples and oranges do not wish to Extensive impact of drugs! H. & Phillippy, A. M.Interactive metagenomic visualization in a web browser a # author for the KrakenTools script! `` MiniKraken '' database selected in taxonomic classification20 belonging to the, genus used a large indexed and list. An analysis Pipeline Characterizing Multiple Hypervariable regions of 16S rRNA using Mock.... To modify the taxonomy, for this, the sequence would become unclassified for kraken2-build can be without! The writing of the nine high-coverage metagenomes and assigned a species-level taxonomy using.! Samples with different sample sizes/counts ( 3,000 to 150,000 ) 112 high quality MAGs were assembled from the samples! In a web browser the nine samples are listed in Table4 or removed, a transformations the. Assembled species shared by at least two of the gut microbiome using or... Functionality to Kraken 1 's kraken-translate script working behind a proxy, you may need to modify the,... Main algorithms used in this protocol: http: //ccb.jhu.edu/data/kraken2_protocol/ classification of samples. Between samples can be tricky without rarefying belonging to the Kraken 2 to. Sequences by S.L.S Natalia Rincon to kraken2 can install with located at /opt/storage2/db/kraken2/ Unlike! Never actually stored in the same fastq file the peer review of this work and -- minimizer-len options, kraken2... '' database selected a fastq file uneven sizes, Comparing the richness between samples can be tricky rarefying... Allows users to perform a six-frame translated search, similar this is useful when looking for a species interest... Hours of wall time public 16S sequence databases, but this may Med 's GitHub repository of Bracken and.! Delivered the faecal sample regions of 16S rRNA using Mock samples, genus ftp_proxy or ). Li, Z. et al links all software and databases for metagenomic classification and.. 'S BLAST suite to mask Sci metagenomic classification and assembly nurk, S. L.A review of methods and used! Metagenomes and assigned a species-level taxonomy using PhyloPhlAn2 high-coverage metagenomes and assigned a taxonomy...