NEXTFLEX^® 16S V1-V3 Amplicon-Seq Kit for Illumina^® Platforms

SEQUENCES

• Sequences of NEXTFLEX 16S V1-V3 Amplicon-Seq Barcoded Primers Indexes – Excel / PDF

APPLICATION NOTE

• Metagenomics Studies of Plant-associated Microbial Communities

Selected Citations that Reference the Use of the NEXTFLEX 16S V1-V3 Amplicon-Seq Kit

• Cortez, V., Canal, E., Dupont-Turkowsky, J. C., Quevedo, T., Albujar, C., Chang, T., . . . Bausch, D. G. (2018). Identification of Leptospira and Bartonella among rodents collected across a habitat disturbance gradient along the Inter-Oceanic Highway in the southern Amazon Basin of Peru. Plos One, 13(10). doi:10.1371/journal.pone.0205068.
• Franzen, J., Zirkel, A., Blake, J., Rath, B., Benes, V., Papantonis, A., & Wagner, W. (2016). Senescence-associated DNA methylation is stochastically acquired in subpopulations of mesenchymal stem cells. Aging Cell, 16(1), 183-191. doi:10.1111/acel.12544.
• Hakim, H., Dallas, R., Wolf, J., Tang, L., Schultz-Cherry, S., Darling, V., . . . Rosch, J. W. (2018). Gut Microbiome Composition Predicts Infection Risk During Chemotherapy in Children With Acute Lymphoblastic Leukemia. Clinical Infectious Diseases,67(4), 541-548. doi:10.1093/cid/ciy153.
• Luna, R. A., Oezguen, N., Balderas, M., Venkatachalam, A., Runge, J. K., Versalovic, J., . . . Williams, K. C. (2017). Distinct Microbiome-Neuroimmune Signatures Correlate With Functional Abdominal Pain in Children With Autism Spectrum Disorder. Cellular and Molecular Gastroenterology and Hepatology, 3(2), 218-230. doi:10.1016/j.jcmgh.2016.11.008.
• Maiuri, A. R., et al. (2017) Mismatch Repair Proteins Initiate Epigenetic Alterations during Inflammation-Driven Tumorigenesis. Cancer Research, 77(13), 3467-3478. doi:10.1158/0008-5472.can-17-0056.
• Quereda, J. J., et al. (2016) Bacteriocin from epidemic Listeria strains alters the host intestinal microbiota to favor infection. PNAS. doi:10.1073/pnas.1523899113.
• Pahwa, R., Balderas, M., Jialal, I., Chen, X., Luna, R. A., & Devaraj, S. (2017). Gut Microbiome and Inflammation: A Study of Diabetic Inflammasome-Knockout Mice. Journal of Diabetes Research,2017, 1-5. doi:10.1155/2017/6519785.
• Ranjan, R., Rani, A., Metwally, A., McGee, H. S. and Perkins D. L. (2015) Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing. Biochem Biophy Res Com. doi:10.1016/j.bbrc.2015.12.083.
• Su, A., Yang, W., Zhao, L., Pei, F., Yuan, B., Zhong, L., . . . Hu, Q. (2018). Flammulina velutipes polysaccharides improve scopolamine-induced learning and memory impairment in mice by modulating gut microbiota composition. Food & Function, 9(3), 1424-1432. doi:10.1039/c7fo01991b.
• Whon, T. W., Chung, W., Lim, M. Y., Song, E., Kim, P. S., Hyun, D., . . . Nam, Y. (2018). The effects of sequencing platforms on phylogenetic resolution in 16 S rRNA gene profiling of human feces. Scientific Data, 5, 180068. doi:10.1038/sdata.2018.68.
• Yao, J. et al. (2016) A Pathogen-Selective Antibiotic Minimizes Disturbance to the Microbiome. Antimicrob. Agents Chemother. 00535-16. doi:10.1128/AAC.00535-16.

The NEXTFLEX 16S V1 -V3 Amplicon-Seq Kit contains enough material to prepare 8, 24, 96 or 192 amplicon-seq libraries from genomic DNA for Illumina^® sequencing. The shelf life of all reagents is 12 months when stored properly. All components can be safely stored at -20°C. This kit is shipped on dry ice.

Outside of novel sequencing technologies that emerge every few years, the ability to multiplex samples is the most critical and revolutionary aspect of next-generation sequencing. Multiplexing allows for acute control of throughput, amplifying the value of obtaining just enough data per sample.

To make multiplexing possible, small arbitrary sequences are incorporated into the sequencing adapters attached to all fragments of a particular sample. These sequences, known as barcodes, allow for post-sequencing processing to bin each fragment by its originating sample.

However, even high-fidelity polymerases used during sequencing reads are invariably prone to introducing errors. These errors are especially costly when landing during the barcode read, preventing proper binning and wasting associated sequencing reads. To alleviate this, the knowledge of bitwise error correction was extended to the base-wise language of sequencing.

The overall ability to correct barcode read errors stems from the differentiability between the entire set of barcodes. Differentiability can be called distance, or the number of single position changes that are required for one barcode sequence to become another. For example, the top sequence in the below figure has only one position change from the middle, while the middle has one position change from the bottom. Overall, the top to bottom sequence requires two position changes. This concept, known as the Hamming distance, is what powers barcode error correction and casual codebreaking games like Mastermind.

The greater the minimum distance separation across an entire barcode set, the stronger the differentiability. This in turn governs how many errors can be error-corrected across a barcode subset. Maximum error correction is governed by the following formula:

where d is the minimum distance across the entire set.

How does minimum distance affect generating barcode sets? By increasing the minimum distance across a subset, the overall maximum subset size decreases. One must set requirements so that sufficient barcodes are within a set of desired error correction.

We have expanded previously available barcode sequence sets in both set size and index lengths to accommodate higher levels of error correction. Also, other factors such as colorspace on Illumina instruments have been considered, leaving customers with a minimal amount of effort in selecting the best subsets for low-diversity sequencing runs.

Our new 12 nt barcode set, available with the NEXTFLEX^® 16S V1 – V3 Amplicon-Seq Kit, allows for up to two error corrections and has multiple low-diversity pooling options. We will continue to develop new technologies to remain the leader in quality multiplexing options.