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Frequently Asked Questions

The most common questions we get asked. If you don’t see the answer to any of your questions please send us a message in the form below.

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Most frequent questions and answers

Our database has quarterly update schedule.

No, the “Identify” service can only upload a 16S rRNA sequence one by one. And it’s free up to 200 IDs only for academia and non-profit institutions. If you want to upload multi-sequences, we recommend you use the commercial ID service, “TrueBac ID-16S” which uses a more stringently validated database.

To learn more about TrueBac ID-16S, please visit here.

EzBioCloud’s 16S database is indeed based on the taxa published on the taxa published in IJSEM. For particular taxa, we assign the representative 16S rRNA sequence based on the TYPE strain. As for individual bacterial isolates, we tend to run quality control by obtaining complete 16S sequence by using multiple rounds of PacBio sequencing. In cases where we see high copy number variants which are also fairly evenly distributed, we usually determine the sample to be contaminated.

In a single cell of bacteria, the 16S gene copy number can vary as much as up to 15 times among bacterial strains. NGS reads obtained from 16S amplicon samples are assigned to known taxa, and the number of 16S reads can be used to calculate the relative taxonomic abundance. However, to get the true relative taxonomic abundance, the number of 16S reads should be normalized by 16S gene copy number and we finally get the number of cells for each species. EzBioCloud includes 16S gene copy number information for over 3,200 species and the predicted 16S copy number by PICRUSt is provided for the remaining species.

To read more about 16S copy number correction, please refer to here.

Yes. EzBioCloud’s “Identify” engine automatically recognizes reverse-complement sequences. They are corrected before being fed to our identification pipeline. We save the corrected sequences in your account, instead of the original version.

Up to ten identifications can be running simultaneously, but they need to be submitted one by one. The ‘Identify single sequence’ button allows the user to submit a single 16S rRNA gene sequence for identification, but the user can sequentially submit additional single sequences for identification, and the system will allow up to 10 identification jobs running simultaneously per user.

For public users, up to 200 results are saved. If a new result is added, the oldest result will be deleted.

Pairwise sequence similarity is the key criterion for microbial identification and as suggested by Kim et al. (2014), a species boundary is defined by a similarity value of 98.65% or greater. In other words, if two sequences show a similarity of 98.65% or less, they belong to different species. BLAST and other services may produce different similarities as they use different algorithms.
BLASTN gives identity value which is match/total length, while EzBiocloud gives similarity value which is match/(match+mismatch). We recommend avoiding BLAST for taxonomy-related sequence identity values.

To learn more about pairwise nucleotide sequence alignment and how to calculate pairwise sequence similarity, please refer to the links.

[Pairwise alignment]
[How to calculate similarity]

Yes, but this function can only be done using the command line tool, OAU. We have an example on our guide here under ‘Multiple ANI calculation example’.

For community users, up to 200 results are saved for 1 year. If 200 IDs were already done and a new result is added, the oldest result will be deleted.

Please watch this video for how to get phylogenetic tree from EzBioCloud “Identify” result.

[Watch video]

Microbiome Taxonomic Profiling(MTP)

Most frequent questions and answers

For EzBioCloud Microbiome Taxonomic Profiling cloud service, up-to 100,000 reads can be processed per each sample. This limit is after QC and merging paired-end reads and only applied to our free trial, BASIC and PREMIUM subscription models. However, our ENTERPRISE model has no limit on the size of raw data [Learn more].

The following demonstration microbiome contains >9 million reads of Illumina MiSeq V3V5 sequencing which was processed by EzBioCloud MTP pipeline.


The different ‘Region’ assignments use different parts of sequences within the database. For example, for Shotgun, MTP uses V1V9 full sequences as the reference database and for V4, MTP uses only the V4 region sequences as the reference database. This situation makes little difference in alignment and similarity. Meaning, in most cases, the profile results are not different. However, sometimes, based on the different similarity, the species-level identified read count(>97% similarity) is changed making a difference in the results when there is nearly 97% similarity.

With a premium account, you can upload up to 10 samples at a time provided the file upload size is smaller than 1gb. If you need to upload larger batch sizes regularly please contact admin@ezbiome.com

With a premium account, you can share your results provided the colleague or client you would like to share with has an EzBioCloud account.

TrueBac ID

Most frequent questions and answers

Bacterial identification is the process of assigning a strain to a known species that was previously classified and named using modern taxonomical principle. In theory, the accuracy of identification is guaranteed if it uses the same method as classification process. At present, bacterial classification is based on the comparison of whole genome sequences, as a form of pairwise genome similarity called Average Nucleotide identity (ANI) [Learn more]. Because TrueBac ID uses the taxonomically verified reference database and scientifically proven algorithm, the results of identification are more accurate than any other technologies. If the genome sequence of an isolate shows >96% ANI value to the taxonomically verified genome sequence of the type strain of Vibrio cholerae,  it is identified as Vibrio cholerae with 100% accuracy, scientifically.

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