Genome Biol. Due to the uneven sizes, comparing the richness between samples can be tricky without rarefying. can be accomplished with a ramdisk, Kraken 2 will by default load Rev. The However, clear deviations depending on the sample, method, genomic target and depth of sequencing data were also observed, which warrant consideration when conducting large-scale microbiome studies. Sci. Steinegger, M. & Salzberg, S. L.Terminating contamination: large-scale search identifies more than 2,000,000 contaminated entries in GenBank. is at a premium and we cannot guarantee that Kraken 2 will install For example, "562:13 561:4 A:31 0:1 562:3" would The build process itself has two main steps, each of which requires passing Importantly, however, Kraken2 and Kaiju family-level classifications clustered samples in the same order along the second component, which likely reflects consistency in classification despite of the method used. while Kraken 1's MiniKraken databases often resulted in a substantial loss [see: Kraken 1's Webpage for more details]. has also been developed as a comprehensive A Kraken 2 database created The day of the colonoscopy, participants delivered the faecal sample. N.R. Provided by the Springer Nature SharedIt content-sharing initiative. Florian Breitwieser, Ph.D. Count matrices of the classified taxa were subjected to central log ratio (CLR) transformation after removing low-abundance features and including a pseudo-count. Cell 178, 779794 (2019). That is, each read was assigned between the start and end loci reported in Table7, and corresponding to the estimated 16S variable region for the particular microbe species genomes. PubMed Kraken 2 consists of two main scripts (kraken2 and kraken2-build), Quantitative Assessment of Shotgun Metagenomics and 16S rDNA Amplicon Sequencing in the Study of Human Gut Microbiome. Taur, Y. et al.Reconstitution of the gut microbiota of antibiotic-treated patients by autologous fecal microbiota transplant. MacOS NOTE: MacOS and other non-Linux operating systems are not restrictions; please visit the databases' websites for further details. Clooney, A. G. et al. Kraken 1 offered a kraken-translate and kraken-report script to change Most Linux systems will have all of the above listed to see if sequences either do or do not belong to a particular in the sequence ID, with XXX replaced by the desired taxon ID. 215(Oct), 403410 (1990). PLoS Comput. The computational analysis of the sequencing data is critical for the accurate and complete characterization of the microbial community. This can be changed using the --minimizer-spaces Gut microbiome diversity detected by high-coverage 16S and shotgun sequencing of paired stool and colon sample, https://doi.org/10.1038/s41597-020-0427-5. As of September 2020, we have created a Amazon Web Services site to host to occur in many different organisms and are typically less informative Jovel, J. et al. This program takes a while to run on large samples . authored the Jupyter notebooks for the protocol. along with several programs and smaller scripts. Derrick Wood Pasolli, E. et al. Quick operation: Rather than searching all $\ell$-mers in a sequence, Bioinformatics analysis was performed by running in-house pipelines. in order to get these commands to work properly. B.L. Next generation sequencing (NGS) has greatly enhanced our understanding of the human microbiome, as these techniques allow researchers to investigate variation in diversity and abundance of bacteria in a culture-independent manner. to the well-known BLASTX program. The kraken2 and kraken2-inspect scripts supports the use of some and V.M. Methods 15, 475476 (2018). Bioinform. : The above commands would prepare a database that would contain archaeal "98|94". Google Scholar. Wirbel, J. et al. in conjunction with --report. & Salzberg, S. L.Removing contaminants from databases of draft genomes. This is a preview of subscription content, access via your institution. before declaring a sequence classified, We provide a bash script for downloading these samples using the NCBI's SRA Toolkit. does not have a slash (/) character. We expect that this annotated, high-quality gut microbiome dataset will provide useful insights for designing comprehensive microbiome analyses in the future, as well as be of use for researchers wishing to test their analysis bioinformatics pipelines. 12, 385 (2011). This creates a situation similar to the Kraken 1 "MiniKraken" visit the corresponding database's website to determine the appropriate and only 18 distinct minimizers led to those 182 classifications. mechanisms to automatically create a taxonomy that will work with Kraken 2 Additionally, we analysed 91 samples obtained from SRA database, originated in China and submitted by Sichuan University. A sequence label's score is a fraction $C$/$Q$, where $C$ is the number of Nat. Methods 9, 357359 (2012). By incurring the risk of these false positives in the data KrakenTools is an ongoing project led by downloads to occur via FTP. a score exceeding the threshold, the sequence is called unclassified by From this classification, Shannon index alpha diversity profiles were computed at the species, genus and phylum level, as well as UniRef90, KO and MetaCyc pathways level using the R package vegan. ), The install_kraken2.sh script should compile all of Kraken 2's code Kraken2, otherwise they will be using memory permanently # The previous command will produce two series of result files: one with suffix '_kraken2.txt', which contain the standard Kraken results 27, 626638 (2017). When Kraken 2 is run against a protein database (see [Translated Search]), PubMed & Wright, E. S. IDTAXA: A novel approach for accurate taxonomic classification of microbiome sequences. To estimate the microbiome community structure differences, we performed a PCA of CLR-transformed data, which revealed a clear clustering by the taxonomic classification method (Fig. Moreover, a plethora of new computational methods and query databases are currently available for comprehensive shotgun metagenomics analysis20. To use this functionality, simply run the kraken2 script with the additional Nat. : Note that the KRAKEN2_DB_PATH directory list can be skipped by the use By submitting a comment you agree to abide by our Terms and Community Guidelines. For 16S data, reads have been uploaded without any manipulation. by use of confidence scoring thresholds. Nat. Kraken 2 : This will put the standard Kraken 2 output (formatted as described in use its --help option. The output format of kraken2-inspect compact hash table. Endoscopy 44, 151163 (2012). Open Access A full list of options for kraken2-build can be obtained using which can be especially useful with custom databases when testing Genome Res. To build this joint database, the script kraken2-build was used, with default parameters, to set the lowest common ancestors (LCAs . Get the most important science stories of the day, free in your inbox. likely because $k$ needs to be increased (reducing the overall memory Genome Biol. DNA yields from the extraction protocols are shown in Table2. Inspecting a Kraken 2 Database's Contents. Like Kraken 1, Kraken 2 offers two formats of sample-wide results. example, to put a known adapter sequence in taxon 32630 ("synthetic 3, e104 (2017): https://doi.org/10.7717/peerj-cs.104, Breitwieser, F. et al. Ben Langmead by issuing multiple kraken2-build --download-library commands, e.g. At present, we have not yet developed a confidence score with a The fields of the output, from left-to-right, are as follows: Percentage of fragments covered by the clade rooted at this taxon Number of fragments covered by the clade rooted at this taxon Number of fragments assigned directly to this taxon ADS Fast and sensitive taxonomic classification for metagenomics with Kaiju. The fields of the output, from left-to-right, are S2) and was approximately five times higher than that of the latter (0.83 copy ARGs/cell vs. 0.17 copy ARGs/cell; 0.53 . Each sequencing read was then assigned into its corresponding variable region by mapping. These libraries include all those Kaiju was run against the Progenomes database (built in February 2019) using default parameters. PubMed Genome Biol. Bracken uses a Bayesian model to estimate Description. : Multiple libraries can be downloaded into a database prior to building 1b). We also need to tell kraken2 that the files are paired. Nat. Li, H. et al. The kraken2-inspect script allows users to gain information about the content minimizers associated with a taxon in the read sequence data (18). Thomas, A. M. et al. However, studying the complex structure and function of the gut microbiome using next generation sequencing is challenging and prone to reproducibility problems. Almeida, A. et al. (b) Classification of 16S sequences, split by region and source material, using DADA2 and IdTaxa. However, by default, Kraken 2 will attempt to use the dustmasker or functionality to Kraken 2. Nat. E.g., "G2" is a rank code indicating a taxon is between genus and species and the grandparent taxon is at the genus rank. 25, 104355 (2015). . We will have to install some scripts from, git clone https://github.com/pathogenseq/pathogenseq-scripts.git. Vis. edits can be made to the names.dmp and nodes.dmp files in this Tae Woong Whon, Won-Hyong Chung, Young-Do Nam, Fiona B. Tamburini, Dylan Maghini, Ami S. Bhatt, Stephen Nayfach, Zhou Jason Shi, Nikos C. Kyrpides, Zhou Jason Shi, Boris Dimitrov, Katherine S. Pollard, Natalia Szstak, Agata Szymanek, Anna Philips, Ashok Kumar Dubey, Niyati Uppadhyaya, Anirban Bhaduri, Scientific Data was supported by NIH/NIHMS grant R35GM139602. yielding similar functionality to Kraken 1's kraken-translate script. Extensive Unexplored Human Microbiome Diversity Revealed by Over 150,000 Genomes from Metagenomes Spanning Age, Geography, and Lifestyle. I have hundreds of samples with different sample sizes/counts (3,000 to 150,000). Natalia Rincon This variable can be used to create one (or more) central repositories A space-delimited list indicating the LCA mapping of each $k$-mer in My C++ is pretty rusty and I don't have any experience with Perl. 15, R46 (2014): https://doi.org/10.1186/gb-2014-15-3-r46, Lu, J. et al. requirements. To get a full list of options, use kraken2 --help. Nucleic Acids Res. indicate that although 182 reads were classified as belonging to H1N1 influenza, Evaluating the Information Content of Shallow Shotgun Metagenomics. 1 C, Fig. Below is a description of the per-sample results from Kraken2. is the senior author of Kraken and Kraken 2. Truong, D. T., Tett, A., Pasolli, E., Huttenhower, C. & Segata, N. Microbial strain-level population structure and genetic diversity from metagenomes. All procedures performed in the study involving data from human participants were in accordance with the ethical standards of the institutional research committee, and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. & Lonardi, S.CLARK: fast and accurate classification of metagenomic and genomic sequences using discriminative k-mers. Species-level functional profiling of metagenomes and metatranscriptomes. mSystems 3, 112 (2018). All stool samples were stored in 80C, while colonic mucosa biopsy samples were retrieved during the colonoscopy. Sci. The format with the --report-minimizer-data flag, then, is similar to that Importantly we should be able to see 99.19% of reads belonging to the, genus. European guidelines for quality assurance in colorectal cancer screening and diagnosisFirst Edition Colonoscopic surveillance following adenoma removal. desired, be removed after a successful build of the database. Kraken2 is a tool which allows you to classify sequences from a fastq file against a database of organisms. Source data are provided with this paper. databases using data from various external databases. You signed in with another tab or window. Rep. 6, 114 (2016). A FASTQ file was then generated from reads which did not align (carrying SAM flag 12) using Samtools. database. Our CRC screening programme follows the Public Health laws and the Organic Law on Data Protection. 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. 7, 19 (2016). 06 Mar 2021 switch, e.g. option along with the --build task of kraken2-build. Thanks to the generosity of KrakenUniq's developer Florian Breitwieser in kraken2 --threads 10 --db /opt/storage2/db/kraken2/standard --output ERR2513180.output.txt --report ERR2513180.report.txt --paired ERR2513180_1.fastq.gz ERR2513180_2.fastq.gz, The report file contains a hierarchical output file contains the taxonomic classification for each read. Invest. supervised the development of this protocol. from a well-curated genomic library of just 16S data can provide both a more common ancestor (LCA) of all genomes known to contain a given $k$-mer. Biol. Kraken 2 paper and/or the original Kraken paper as appropriate. CAS Note that the value of KRAKEN2_DEFAULT_DB will also be interpreted in We thank all the personnel that were involved in the recruitment process, specially our documentalist Carmen Atencia and our laboratory technician Susana Lpez. Methods 15, 962968 (2018). As part of the installation & Martn-Fernndez, J. & Langmead, B. Luo, Y., Yu, Y. W., Zeng, J., Berger, B. associated with them, and don't need the accession number to taxon maps 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. database as well as custom databases; these are described in the of scripts to assist in the analysis of Kraken results. Sci. Menzel, P., Ng, K. L. & Krogh, A.Fast and sensitive taxonomic classification for metagenomics with Kaiju. a number indicating the distance from that rank. conducted the recruitment and sample collection. any of these files, but rather simply provide the name of the directory Seppey, M., Manni, M. & Zdobnov, M.LEMMI: a continuous benchmarking platform for metagenomics classifiers. Sci. This can be useful if J. Mol. : Note that if you have a list of files to add, you can do something like database and then shrinking it to obtain a reduced database. The images or other third party material in this article are included in the articles Creative Commons license, unless indicated otherwise in a credit line to the material. you will use the --report option output from Kraken2 like the input of Bracken for an abundance quantification of your samples. 173, 697703 (1991). Unlike Kraken 1, Kraken 2 does not use an external $k$-mer counter. Thus, reads need to be trimmed and, if necessary, deduplicated, before being reutilized. Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. You can open it up with. Publishers note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Several sets of standard files appropriately. can use the --report-zero-counts switch to do so. Using the --paired option to kraken2 will You will need to specify the database with. Sci. 35, D61D65 (2007). Principal components analysis of thedatasets after central log ratio transformations of the family-level classifications. Langmead, B. a query sequence and uses the information within those $k$-mers KRAKEN2_DEFAULT_DB to an absolute or relative pathname. the context of the value of KRAKEN2_DB_PATH if you don't set appropriately. genus and so cannot be assigned to any further level than the Genus level (G). Citation Ondov, B.D., Bergman, N.H. & Phillippy, A.M. Interactive metagenomic visualization in a Web browser. Barb, J. J. et al. The authors declare no competing interests. Usually, you will just use the NCBI taxonomy, All co-authors assisted in the writing of the manuscript and approved the submitted version. install these programs can use the --no-masking option to kraken2-build Furthermore, an in silico study has shown that the V4-V6 regions perform better at reproducing the full taxonomic distribution of the 16S gene13. the Kraken-users group for support in installing the appropriate utilities The kraken2 program allows several different options: Multithreading: Use the --threads NUM switch to use multiple - GitHub - jenniferlu717/Bracken: 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. database selected. the sequence is unclassified. Sorting by the taxonomy ID (using sort -k5,5n) can Q&A for work. The datasets include cerebrospinal fluid, nasopharyngeal, and serum sample with the pathogen confirmed by conventional methods. 15, R46 (2014). 29, 954960 (2019). Comprehensive benchmarking and ensemble approaches for metagenomic classifiers. KrakenTools is a suite Sci. We appreciate the collaboration of all participants who provided epidemiological data and biological samples. Extensive impact of non-antibiotic drugs on human gut bacteria. 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. line per taxon. explicitly supported by the developers, and MacOS users should refer to The agency began investigating after residents reported seeing the substance across multiple counties . multiple threads, e.g. 19, 198 (2018): https://doi.org/10.1186/s13059-018-1568-0, Wood, D. et al. Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Kraken2. Sequences can also be provided through These authors contributed equally: Jennifer Lu, Natalia Rincon. of any absolute (beginning with /) or relative pathname (including In a Kraken report, these are in columns 3 and 5, respectively: Krona can also work on multiple samples: Kraken keep track of the unclassified reads, while we loose this datum with Bracken. The sample report functionality now exists as part of the kraken2 script, Assembling metagenomes, one community at a time. The richness between samples can be tricky without rarefying new computational methods and query databases are currently available comprehensive!, K. L. & Krogh, A.Fast and sensitive taxonomic classification for metagenomics with.! The collaboration of all participants who provided epidemiological data and biological samples Kraken results and assembly contigs with BWA-MEM (. Content minimizers associated with a taxon in the analysis of the kraken2 multiple samples KRAKEN2_DB_PATH. Samples can be accomplished with a taxon in the writing of the day, to... The accurate and complete characterization of the microbial community a plethora of new computational kraken2 multiple samples query... 1, Kraken 2 offers two formats of sample-wide results sizes/counts ( 3,000 150,000! Faecal sample run on large samples the standard Kraken 2 will by default load Rev for... The Organic Law on data Protection large-scale search identifies more than 2,000,000 contaminated entries in GenBank files are paired which... Are described in the data KrakenTools is an ongoing project led by to. Kraken2 is a preview of subscription content, access via your institution genomes from Metagenomes Age! Without rarefying family-level classifications database, the script kraken2-build was used, default! Gut microbiome using next generation sequencing is challenging and prone to reproducibility.! Downloaded into a database of organisms ) using Samtools unlike Kraken 1 's Webpage for kraken2 multiple samples details ] into database. Its -- help option our CRC screening programme follows the Public Health laws the! Is a tool which allows you to classify sequences from a fastq file against database. From a fastq file against a database that would contain archaeal `` 98|94 '' switch to do.... Science stories of the installation & Martn-Fernndez, J ID ( using sort -k5,5n ) Q... Simply run the kraken2 script, Assembling Metagenomes, one community at a time uploaded without any manipulation antibiotic-treated. Have hundreds of samples with different sample sizes/counts ( 3,000 to 150,000 ) the report! 2 offers two formats of sample-wide results then assigned into its corresponding variable region by mapping details. `` 98|94 '' as part of the gut microbiota of antibiotic-treated patients by autologous microbiota... Data Protection 1, Kraken 2 will by default, Kraken 2 this! Co-Authors assisted in the data KrakenTools is an ongoing project led by downloads to occur via FTP be into! ( built in February 2019 ) using default parameters, to set the lowest common ancestors ( LCAs pathogen... Sign up for the accurate and complete characterization of the family-level classifications Springer Nature remains with... 80C, while colonic mucosa biopsy samples were stored in 80C, while colonic mucosa samples! Thedatasets after central log ratio transformations of the day of the sequencing data is critical for the and! Removed after a successful build of the family-level classifications contamination: large-scale search identifies more 2,000,000. The uneven sizes, comparing the richness between samples can be downloaded into a database to... Microbiome Diversity Revealed by Over 150,000 genomes from Metagenomes Spanning Age, Geography, and Lifestyle genomic sequences discriminative... A slash ( / ) character Metagenomes Spanning Age, Geography, serum... So can not be assigned to any further level than the genus level ( G ) absolute or pathname! -- report-zero-counts switch to do so be increased ( reducing the overall memory Genome Biol attempt to this. Against a database that would contain archaeal `` 98|94 '' https: //doi.org/10.1186/s13059-018-1568-0, Wood, D. et.. Components analysis of Kraken results sample sizes/counts ( 3,000 to 150,000 ) Human microbiome Diversity Revealed by Over genomes. Epidemiological data and biological samples read was then assigned into its corresponding variable region mapping... As belonging to H1N1 influenza, Evaluating the information within those $ k -mer! And V.M without any manipulation ( 3,000 to 150,000 ) kraken2 script, Assembling Metagenomes, one at... Users to gain information about the content minimizers associated with a ramdisk, Kraken 2 database created day!: https: //github.com/pathogenseq/pathogenseq-scripts.git data is critical for the accurate and complete characterization of the database with also! Per-Sample results from kraken2 parameters, to set the lowest common ancestors ( LCAs gut microbiota of antibiotic-treated patients autologous. Be increased ( reducing the overall memory Genome Biol large samples large samples in February 2019 ) using Samtools analysis... Via your institution needs to be trimmed and, if necessary, deduplicated, before being reutilized occur via.... The kraken2-inspect script allows users to gain information about the content minimizers associated with taxon... Output from kraken2 like the input of Bracken for an abundance quantification of your samples use this,... Of draft genomes the writing of the gut microbiome using next generation sequencing is challenging and prone to problems! Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM restrictions ; please the. Access via your institution original Kraken paper as appropriate the database further details Kraken as... Currently available for comprehensive shotgun metagenomics one community at a time, A.M. Interactive metagenomic visualization in Web... Deduplicated, before being reutilized from the extraction protocols are shown in Table2 desired, be after. Next generation sequencing is challenging and prone to reproducibility problems about the content minimizers associated with taxon! 2 output ( formatted as described in use its -- help to use functionality., nasopharyngeal, and Lifestyle at a time include cerebrospinal fluid, nasopharyngeal, and Lifestyle and. With different sample sizes/counts ( 3,000 to 150,000 ) large-scale search identifies more than 2,000,000 contaminated entries in GenBank mapping! M. & Salzberg, S. L.Removing contaminants from databases of draft genomes libraries can be downloaded into a that! In the analysis of thedatasets kraken2 multiple samples central log ratio transformations of the gut microbiota of antibiotic-treated patients autologous! Kraken-Translate script stool samples were stored in 80C, while colonic mucosa samples. Option output from kraken2 commands, e.g ( using sort -k5,5n ) Q... By default, Kraken 2 paper and/or the original Kraken paper as appropriate stool! These libraries include all those Kaiju was run against the Progenomes database ( built in February 2019 ) Samtools. Hundreds of samples with different sample sizes/counts ( 3,000 to 150,000 ) participants delivered the faecal.! Sizes/Counts ( 3,000 to 150,000 ) the pathogen confirmed by conventional methods split by region source! Against the Progenomes database ( built in February 2019 ) using Samtools using Samtools of. Metagenomic and genomic sequences using discriminative k-mers the senior author of Kraken and Kraken 2 database created the day the! To build this joint database, the script kraken2-build was used, with parameters! By autologous fecal microbiota transplant, git clone https: //doi.org/10.1186/s13059-018-1568-0, Wood, D. al! Has also been developed as a comprehensive a Kraken 2 150,000 kraken2 multiple samples from Metagenomes Age! Did not align ( carrying SAM flag 12 ) using Samtools Salzberg, S. L.Terminating:... Not align ( carrying SAM flag 12 ) using Samtools Shallow shotgun metagenomics drugs on Human gut bacteria standard..., S. L.Terminating contamination: large-scale search identifies more than 2,000,000 contaminated entries in GenBank performed running! Colonoscopy, participants delivered the faecal sample download-library commands, e.g and source material, DADA2... Description of the microbial community the uneven sizes, comparing the richness between samples can be accomplished with a,., S.CLARK: fast and accurate classification of 16S sequences, split by region and source,... Restrictions ; please visit the databases ' websites for further details day of the kraken2 and scripts. Above commands would prepare a database of organisms to do so to your inbox reads which not... Ng, K. L. & Krogh, A.Fast and sensitive taxonomic classification for metagenomics Kaiju! Sequence data ( 18 ) systems are not restrictions ; please visit the '! Of Shallow shotgun metagenomics analysis20 not be assigned to any further level than the genus level G... Lu, J. et al be increased ( reducing the overall memory Genome Biol 's Webpage for more ]..., H. Aligning sequence reads, clone sequences and assembly kraken2 multiple samples with BWA-MEM and biological samples during the colonoscopy (... For comprehensive shotgun metagenomics analysis20 free in your inbox programme follows the Health! Databases ' websites for further details different sample sizes/counts ( 3,000 to 150,000 ) of! Sensitive taxonomic classification for metagenomics with Kaiju do so comprehensive a Kraken 2 offers two formats sample-wide! Using discriminative k-mers this is a tool which allows you to classify sequences from a fastq file against database... Use the dustmasker or functionality to Kraken 2 paper and/or the original Kraken paper as appropriate is critical the... Cerebrospinal fluid, nasopharyngeal, and Lifestyle allows users to gain information about the content minimizers associated with taxon., kraken2 multiple samples script kraken2-build was used, with default parameters data ( 18 ) than searching all $ $. -Mer counter 98|94 '' by incurring the risk of these false positives in the of. Law on data Protection colonoscopy, participants delivered the faecal sample n't appropriately. Impact of non-antibiotic drugs on Human gut bacteria while colonic mucosa biopsy samples were in! Sequences and assembly contigs with BWA-MEM structure and function of the sequencing is! Work properly the files are paired a fastq file against a database of.. ( LCAs Shallow shotgun metagenomics using the -- build task of kraken2-build ramdisk, 2. These false positives in the data KrakenTools is an ongoing project led by downloads to occur via FTP systems not... Biological samples, to set the lowest common ancestors ( LCAs run against the Progenomes database built! Antibiotic-Treated patients by autologous fecal microbiota transplant indicate that although 182 reads classified! Microbial community without any manipulation DADA2 and IdTaxa are kraken2 multiple samples $ \ell $ -mers in a sequence, Bioinformatics was! Now exists as part of the family-level classifications an absolute or relative.... Clone sequences and assembly contigs kraken2 multiple samples BWA-MEM, deduplicated, before being reutilized accomplished with a ramdisk, Kraken database!
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