Comparison of the sequencing bias of currently available. C table1 exampleofapplicationsfordifferentnexterakits nexterafc11031 nexteraxtfc11096 largecomplexgenomes smallgenomes,amplicons,plasmids. 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. 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 kit sequence small genomes. The fast and simple protocol allows for treatment of one or a pool of libraries just prior to. Laboratory standard operating procedure for pulsenet nextera. We validate this protocol using a nonmodel eukaryotic genome, and. This method can be used for any targeted amplicon sequencing. If you used another index format, you will need to use a different entry for library prep kit.
Illumina nextera xt sample preparation protocol chmi services. Nextera dna flex library preparation now you can use human blood, saliva, and bacterial samples directly in your wholegenome sequencing library preparation. Nextera xt dna sample preparation kit illumina, inc. A total of 1 ng dna input into the nextera xt reaction was targeted for every sample diluted with 10 mm trishcl ph 8. Nextera dna flex library prep kit flexibility for many. The i5 index names vary for different nextera products as follows. Illumina targeted sequencing panels facilitate clinical cancer research by providing expertdefined gene content plus simple data analysis and reporting options. Nextgeneration sequencing sample prep methods beckman. In cases where ndna may not be suitable for analysis i. Illumina nextera xt sample preparation protocol chmi. 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. 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. Dna is quantified using a fluorometricbased method and diluted to 0. The simple modification of illuminas nextera xt protocol significantly decreases the overall cost, while providing comparable, or even better performance.
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. Can anyone help with pcr amplicon sequencing illumina. By utilizing the echo 525 liquid handler with the nextera xt kit, we perform the reactions at a miniaturized scale that reduces excessive. Note that sequencing nextera xt libraries on certain instruments the hiseq 2000, hiseq. This document lists the index adapter sequences for illumina library prep kits. Protocol for nextera dna sample prep kit illuminacompatible. As with any enzymatic system, there is a slight bias in the reaction.
This file can then be emailed or uploaded to asana. Any additional reagents supplied with the original nextera xt kit are not necessary for this protocol and may be discarded. This protocol is for the submission of dna for the generation of pairedend libraries for sequencing on the illumina miseq platform. A performance evaluation of nextera xt and kapa hyperplus. Each kit component must pass rigorous quality control standards, and. A condensed version of the nextera xt dna protocol for experienced users. Nextera xt dna library prep reference guide assess dna purity uv absorbanceis a common methodusedfor assessing the purity of a dna sample. A scaleddown workflow for illumina shotgun sequencing. For reference see agilents accessories catalog for the bravo. Our vast portfolio of biomekautomated nextgeneration sequencing sample prep methods are demonstrated to generate quality data using realworld samples, and includes a growing. 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.
Prepare sequencing libraries for small genomes, pcr amplicons, plasmids, or cdna in as little as 90 minutes, with a low dna input requirement. D september 2014 addedinfofornewindexkitsthatenablepreparationofupto384 indexedpairedendlibraries. Nextgeneration sequencing sample prep methods beckman coulter. 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. Sureselect target enrichment system for illumina paired. Forensic dna casework principally relies on the analysis of short tandem repeats strs from nuclear dna ndna. The level of bias introduced is dependent on the level of gc content. We addressed the identified limitations to develop a robust workflow that supports both rapid and highthroughput. A performance evaluation of nextera xt and kapa hyperplus for. To ensure high accuracy and precision, most library preparation protocols recommend volumes that are within the range of manual pipettes or large volume.
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 protocol and corresponding smarter apollo script described in this user manual have been specifically validated using a 10pg input of total rna at an input volume of 7 l, followed by sequencing library construction from the resulting cdna with the nextera xt library preparation kit. Sureselect target enrichment system for illumina pairedend. To describe the standardized laboratory protocol for whole genome sequencing of bacterial organisms on the miseq benchtop sequencer. Summary nextera xt dna library preparation kits are ideal for experiments where speed and ease are of paramount importance. Protocol for submitting dna for illumina sequencing. Preparing whole genome human mitochondrial dna libraries. 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. Library preparation for small genomes bacteria, archaea, viruses, amplicons, and plasmids in less than 90 minutes.
Save your tapestation results by going to file create report save as pdf. Note that sequencing nextera xt libraries on certain instruments the hiseq 2000, hiseq 15002500 in. The nextera xt protocol is optimized for 1 ng of input dna. The nexteraxt dna library preparation kit uses an engineered transposon to fragment and tagment input dna in a single reaction. Consumable supplier 10lpipettetips generallabsupplier 10lmultichannelpipettes generallabsupplier 10lsinglechannelpipettes generallabsupplier. Select your library prep kit based on the index format used.
The fast and simple protocol allows for treatment of one or a pool of libraries just prior to sequencing on any illumina platform. Target enrichment system for illumina pairedend multiplexed sequencing library protocol. Nextera pcr mm npm 15ul dna 25ul total volume 50ul 10. The sequences are grouped into sections for ampliseq for. Nebnext ultra dna library prep kit for illumina neb. Can anyone help with pcr amplicon sequencing illumina miseq. The ratio of absorbance at 260 nm to absorbanceat 280 nm provides an indication of sample purity. The transposase used in the nextera system carries mutations and is used under conditions that result in nearrandom integration.
Hyperplus libraries were prepared in halfvolume reactions with 5. The nextera xt index kit v2 is available in sets ad. The 19bp transposon dna sequence is present at the 5. Epicentre nextera dna sample prep kit illuminacompatible page 4 6. 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. If you used our plate indexes select idtilmn nextera ud index set a for nextera xt. With the introduction of the nextera xt index kit v2, catalog no.
We addressed the identified limitations to develop a robust workflow that. Nextera xt dna library preparation kit and nextera xt index kit. Protocol for submitting dna for illumina sequencing genomes. Furthermore, our procedure takes less than 5 hours for 96 samples. Because each nextera protocol is designed for a distinct application, it is important to tailor the library validation and clustering.
A scaleddown workflow for illumina shotgun sequencing library preparation. Inexpensive multiplexed library preparation for megabase. Application guide nextera dna library preparation on the ngs express perkinelmer 4 introduction the ngs express system is a compact, easy. Improved workflows for high throughput library preparation. Nextera xt libraries were prepared in accordance with the manufacturers protocol.
Ga09115, ga091120, ga091150, ga091196, and gabc0950 the nextera dna sample prep kit is designed. Fc11002 has been obsolesced nextera xt index kit 96 indexes, 384 samples. 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. Instructions extracted from the ngi stockholmscilifelab sop are available as a pdf file license. Instructions for preparing samples using the nextera xt dna sample preparation kit. Protocol for submitting dna for illumina sequencing genomes nextera xt library prep kit version. 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. Automated nextgeneration sequencing sample preparation methods we partner with customers and leading technology vendors to develop and deliver the best solutions. For the base pair length, we usually use the value of the peak identified by the tapestation analysis software. Laboratory standard operating procedure for pulsenet. Product documentationavailable for download in pdf from the illumina website. Direct bacterial colony sequencing with the nextera dna flex library preparation kit. Nextera xt dna library prep kit reference guide 15031942. Smartseq v4 reagent kit for the smarter apollo system.
N50xnextera dna s50xnextera xt e50xnextera enrichment and nextera rapid capture. C table1 exampleofapplicationsfordifferentnexterakits nextera fc11031 nexteraxtfc11096 largecomplexgenomes smallgenomes,amplicons,plasmids. If you used tube indexes select nextera xt v2 set a, b, c, or d. To describe the standardized laboratory protocol for whole genome sequencing of bacterial organisms on the miseq. Nextera mate pair sample preparation guide 15035209 d. Nextera library validation and cluster density optimization. Removed obsolesced nextera xt index kit 96 indexes, 384 samples. Highspeed, multiplexed 16s amplicon sequencing on the. Please note that adaptors and primers are not included in the kit and are available separately. 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. The flex lysis kit is a separate product that complements the blood dna extraction aspect of the nextera flex library prep kit. 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.
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. Illuminas nextera xt dna kit is a single well protocol that prepares pairedend libraries from sample input types such. Hi all, ive just found in nextera dna sample prep kit illuminacompatible transposon end sequence. 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. 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. 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. Lab sop for pulsenet nextera xt library preperation. 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. Here we propose a further modification of the nextera xt protocol, which uses 120thscale reactions figure 1. I have three amplicons of 300 bp average 1 prokaryotic and 2 eukaryotic.
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