Illumina nextera xt sample preparation protocol chmi services. The simple modification of illuminas nextera xt protocol significantly decreases the overall cost, while providing comparable, or even better performance. Save your tapestation results by going to file create report save as pdf. This document lists the index adapter sequences for illumina library prep kits. By systematic comparison of currently available library preparation kits for illumina sequencing, we demonstrated that strong sequencing bias is introduced in lowgc regions by the nextera xt kit. We validate this protocol using a nonmodel eukaryotic genome, and. The nebnext ultra dna library prep kit for illumina contains reagents for preparation of libraries for nextgeneration sequencing on the illumina platform from 5 ng 1 g input dna, in a streamlined workflow. This protocol is intended for high throughput preparation of 96 ngs libraries in parallel, which requires the 96reaction size of the nextera xt dna library preparation k it illumina, cat.
This file can then be emailed or uploaded to asana. Nebnext ultra dna library prep kit for illumina neb. We addressed the identified limitations to develop a robust workflow that. Illumina targeted sequencing panels facilitate clinical cancer research by providing expertdefined gene content plus simple data analysis and reporting options. To provide the pulsenet participants with a single protocol for performing nextera xt library prep and miseq run setup, thus ensuring interlaboratory comparability of the generated results. Nextgeneration sequencing sample prep methods beckman. The fast and simple protocol allows for treatment of one or a pool of libraries just prior to. Illumina nextera xt sample preparation protocol chmi. Instructions extracted from the ngi stockholmscilifelab sop are available as a pdf file license. If you used tube indexes select nextera xt v2 set a, b, c, or d.
We addressed the identified limitations to develop a robust workflow that supports both rapid and highthroughput. The illumina free adapter blocking reagent is an optional reagent that can be used to treat most types of sequencing libraries to reduce index hopping levels. Automated nextgeneration sequencing sample preparation methods we partner with customers and leading technology vendors to develop and deliver the best solutions. As with any enzymatic system, there is a slight bias in the reaction. The fast and simple protocol allows for treatment of one or a pool of libraries just prior to sequencing on any illumina platform. Comparison of the sequencing bias of currently available. The i5 index names vary for different nextera products as follows. A performance evaluation of nextera xt and kapa hyperplus. If you used our plate indexes select idtilmn nextera ud index set a for nextera xt. Library preparation for small genomes bacteria, archaea, viruses, amplicons, and plasmids in less than 90 minutes. Laboratory standard operating procedure for pulsenet nextera. With the aid of a liquid handling robot, the method described here allows the production of 2x384 samples in a day at a cost comparable to or lower than alternative methods. For wholegenome sequencing, the nextera dna flex library prep kit is the recommended replacement for the nextera dna library prep kit, which has been discontinued. Please note that adaptors and primers are not included in the kit and are available separately.
Nextera xt index kit v2, set c 96 indexes, 384 samples fc12003 nextera xt index kit v2, set d 96 indexes, 384 samples fc12004 sequencing primer kits are required for all sequencers except the miseq system. The ratio of absorbance at 260 nm to absorbanceat 280 nm provides an indication of sample purity. We then tested this miniaturized method on a diverse variety of microbes to show that the method is effective across samples. Nextera xt dna library prep reference guide assess dna purity uv absorbanceis a common methodusedfor assessing the purity of a dna sample. N50xnextera dna s50xnextera xt e50xnextera enrichment and nextera rapid capture.
The transposase used in the nextera system carries mutations and is used under conditions that result in nearrandom integration. Illuminas nextera xt dna kit is a single well protocol that prepares pairedend libraries from sample input types such. A total of 1 ng dna input into the nextera xt reaction was targeted for every sample diluted with 10 mm trishcl ph 8. The nextera xt index kit v2 is available in sets ad. The flex lysis kit is a separate product that complements the blood dna extraction aspect of the nextera flex library prep kit. A scaleddown workflow for illumina shotgun sequencing. The nextera xt protocol is optimized for 1 ng of input dna. To ensure high accuracy and precision, most library preparation protocols recommend volumes that are within the range of manual pipettes or large volume. In cases where ndna may not be suitable for analysis i. Nextera library validation and cluster density optimization. Nextera dna flex library prep kit flexibility for many.
Hyperplus libraries were prepared in halfvolume reactions with 5. Because each nextera protocol is designed for a distinct application, it is important to tailor the library validation and clustering. Our vast portfolio of biomekautomated nextgeneration sequencing sample prep methods are demonstrated to generate quality data using realworld samples, and includes a growing. Target enrichment system for illumina pairedend multiplexed sequencing library protocol. Note that sequencing nextera xt libraries on certain instruments the hiseq 2000, hiseq. Prepare sequencing libraries for small genomes, pcr amplicons, plasmids, or cdna in as little as 90 minutes, with a low dna input requirement.
Product documentationavailable for download in pdf from the illumina website. Nextera xt libraries were prepared in accordance with the manufacturers protocol. By utilizing the echo 525 liquid handler with the nextera xt kit, we perform the reactions at a miniaturized scale that reduces excessive. Sureselect target enrichment system for illumina paired.
If you used another index format, you will need to use a different entry for library prep kit. With the introduction of the nextera xt index kit v2, catalog no. To describe the standardized laboratory protocol for whole genome sequencing of bacterial organisms on the miseq benchtop sequencer. Each kit component must pass rigorous quality control standards, and. Furthermore, our procedure takes less than 5 hours for 96 samples. Each nextera dna flex library prep kit requires one of the following nextera dna flex index kits to complete the protocol, regardless of the sample pooling level used for sequencing. Protocol for submitting dna for illumina sequencing. A condensed version of the nextera xt dna protocol for experienced users. A performance evaluation of nextera xt and kapa hyperplus for. For the base pair length, we usually use the value of the peak identified by the tapestation analysis software. Our protocol reduces the cost per sample 4fold from standard nextera, 3fold from illumina truseq and by almost half compared to the nextera xt kit.
Nextera xt dna library preparation kit prepare sequencing libraries for small genomes, pcr amplicons, plasmids, or cdna in as little as 90 minutes, with a low dna input requirement. Instructions for preparing samples using the nextera xt dna sample preparation kit. The nexteraxt dna library preparation kit uses an engineered transposon to fragment and tagment input dna in a single reaction. Summary nextera xt dna library preparation kits are ideal for experiments where speed and ease are of paramount importance. Nextgeneration sequencing sample prep methods beckman coulter. A scaleddown workflow for illumina shotgun sequencing library preparation. Can anyone help with pcr amplicon sequencing illumina miseq. Forensic dna casework principally relies on the analysis of short tandem repeats strs from nuclear dna ndna. Each kit in the nextera family of library preparation dna, xt dna, custom enrichment, and exome enrichment requires different considerations for final library validation and optimization of cluster density. C table1 exampleofapplicationsfordifferentnexterakits nextera fc11031 nexteraxtfc11096 largecomplexgenomes smallgenomes,amplicons,plasmids. The level of bias introduced is dependent on the level of gc content. I have three amplicons of 300 bp average 1 prokaryotic and 2 eukaryotic. Nextera xt dna sample preparation kit illumina, inc.
This protocol is for the submission of dna for the generation of pairedend libraries for sequencing on the illumina miseq platform. Dna is quantified using a fluorometricbased method and diluted to 0. Epicentre nextera dna sample prep kit illuminacompatible page 4 6. Nextera xt dna library preparation kit and nextera xt index kit. Protocol for nextera dna sample prep kit illuminacompatible.
Sureselect target enrichment system for illumina pairedend. Illumina nextera xt based workflow are constrained by the minimum working volumes needed for manual processing, and once processed, only a small fraction of the library generated is used for the sequencing run. Improved workflows for high throughput library preparation. Preparing whole genome human mitochondrial dna libraries. Nextera pcr mm npm 15ul dna 25ul total volume 50ul 10. Ga09115, ga091120, ga091150, ga091196, and gabc0950 the nextera dna sample prep kit is designed. For reference see agilents accessories catalog for the bravo. This method can be used for any targeted amplicon sequencing. Nov 20, 20 the nextera protocol, which utilises a transposome based approach to create libraries for illumina sequencing, requires pure dna template, an accurate assessment of input concentration and a column cleanup that limits its applicability for highthroughput sample preparation. Can anyone help with pcr amplicon sequencing illumina. Note that sequencing nextera xt libraries on certain instruments the hiseq 2000, hiseq 15002500 in.
The sequences are grouped into sections for ampliseq for. Removed obsolesced nextera xt index kit 96 indexes, 384 samples. Select your library prep kit based on the index format used. Inexpensive multiplexed library preparation for megabase. Protocol for submitting dna for illumina sequencing genomes. The 19bp transposon dna sequence is present at the 5. Nextera xt for pcr amplicons quick workflow for long or short range pcr pcr amp pool amplicons, nextera xt pool samples 90 min total 15 min hands on 7 exons 255416 bp range 12 samples 2x36 6. To describe the standardized laboratory protocol for whole genome sequencing of bacterial organisms on the miseq. C table1 exampleofapplicationsfordifferentnexterakits nexterafc11031 nexteraxtfc11096 largecomplexgenomes smallgenomes,amplicons,plasmids. Fc11002 has been obsolesced nextera xt index kit 96 indexes, 384 samples. Nextera mate pair sample preparation guide 15035209 d. Nextera xt dna library prep kit reference guide 15031942. Highspeed, multiplexed 16s amplicon sequencing on the.
Application guide nextera dna library preparation on the ngs express perkinelmer 4 introduction the ngs express system is a compact, easy. Smartseq v4 reagent kit for the smarter apollo system. Learn how you can take advantage of rapid sample processing, high data quality with low bias, and low sample input requirements with the nextera dna flex library preparation kit on page 5. Consumable supplier 10lpipettetips generallabsupplier 10lmultichannelpipettes generallabsupplier 10lsinglechannelpipettes generallabsupplier. Protocol for submitting dna for illumina sequencing genomes nextera xt library prep kit version. Laboratory standard operating procedure for pulsenet. Any additional reagents supplied with the original nextera xt kit are not necessary for this protocol and may be discarded. This guide contains everything a firsttime user needs, including an overview of the nextera xt dna library prep protocol, tips and techniques, protocol steps, kit contents, and usersupplied consumables. D september 2014 addedinfofornewindexkitsthatenablepreparationofupto384 indexedpairedendlibraries. Nextera xt dna library prep kit sequence small genomes. Direct bacterial colony sequencing with the nextera dna flex library preparation kit.