NGS Library Preparation Support—Getting Started
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Ion AmpliSeq technology offers simple and fast library construction for affordable targeted sequencing of specific human genes or genomic regions. Based on ultrahigh-multiplex PCR, Ion AmpliSeq™ technology requires as little as 10 ng of input DNA to target sets of genes, making sequencing of FFPE samples routine on Ion PGM™ Systems.
A DNA fragment library is constructed from whole genomic DNA and is commonly used for whole-genome resequencing or de novo sequencing. Briefly, the whole genomic DNA is fragmented or sheared, ligated with Ion-specific adapter sequences, and then size-selected for the library fragments of the desired length.
Amplicon libraries are constructed from PCR-amplified DNA fragments and are used for targeted sequencing (e.g., investigating variants at known genomic locations). There are two types of amplicon libraries: short and long.
A short amplicon library contains DNA fragments (targets) with lengths that are compatible with the Ion template preparation kits without any further shearing or fragmentation during library preparation. Additionally, no size selection step is required, as the amplicons are already within the desired size range.
A long amplicon library contains DNA fragments (targets) with lengths that are longer than those compatible with the Ion template kits, and it requires further shearing or fragmentation during library preparation. The library preparation protocol for long amplicons is similar to fragment libraries.
We recommend using the Ion Torrent™ Ion Xpress™ Plus Fragment Library Kit (Cat. No. 4471269) for preparing libraries using the Ion Shear™ enzymatic shearing method, or the Ion Plus Fragment Library Kit (Cat. No. 4471252) if using physical fragmentation. The protocol for both library kits is included in the Ion Xpress Plus gDNA Fragment Library Preparation User Guide. Please also refer to the Decision Tree for DNA Sequencing on the Ion PGM™ System for more information.
Please visit the RNA Sequencing applications space for information regarding sequencing data analysis, including white papers, training videos for Ion Proton transcriptome data analysis, and information about third-party software solutions (i.e., Partek Inc.).
16S is a component of the 30S small subunit of a prokaryotic ribosome. It is a ~1500bp region that can be used to identify and classify microbes.
The Ion 16S™ Metagenomics Kit uses two primer sets to selectively amplify the corresponding hypervariable regions of the 16S rDNA gene in bacterial samples:
- 16S Primer Set V2-4-8
- 16S Primer Set V3-6,7-9.
The Ion 16S™ Metagenomics Kit uses TaqMan™ Environmental Master Mix 2.0.
Yes, the TaqMan™ Environmental Master Mix 2.0 is available separately as Cat. No. 4396838.
The Ion 16S™ Metagenomics Kit includes E. coli control DNA (strain K-12).
We recommend using the Ion Universal Library Quantitation Kit, Cat. No. A26217. It is compatible with the uracil-containing amplicons generated by the Ion 16S™ Metagenomics Kit.
The workflow, which consists of library generation, template prep, sequencing, and analysis, takes 2 days.
Use the 16S Metagenomics workflow in Ion Reporter.
The 16S Metagonics Ion Reporter workflow uses the MicroSeq ID and GreenGenes databases.
The data can be analyzed in less than 1 hour.
With this kit, you can generate inserts between 2-8 kb.
We recommend that you start with 2-10 µg of gDNA.
The Ion TrueMate™ Plus Library Kit includes a selection of restriction enzymes needed for the generation of mate-pair libraries. If you would like to select your own restriction enzymes for optimal cleavage of your particular genome, please use the Ion TrueMate™ Library Kit.
No, Ion TrueMate™ libraries are ~400 bp long, so they are only compatible with 400 bp sequencing on the Ion PGM™ System.
No, only mechanical shearing using Diagenode’s Megaruptor™ system and Covaris instrument have been tested.
It takes 3 days to complete the protocol.
Customers with bioinformatics expertise can use SPAdes v3.1.1 plugin on the Ion Torrent™ server or DNASTAR™ Lasergene™ Genomics Suite software. For users who are new to bioinformatics, we offer Field Bioinformatics Scientist (FBS) De Novo Assembly Services, Cat. No. A26190, which is a fully outsourced bioinformatics solution for help with data analysis.
SPAdes v3.1.1 is a command line-based analysis program, and is recommend for customers with a high level of bioinformatics expertise. DNASTAR™ Lasergene™ Genomics Suite software is an analysis program that utilizes an easy-to-use user interface, and comes with commercial support. It is recommended for users with a medium level of bioinformatics expertise.
It is Field Bioinformatics Scientist De Novo Assembly Services (Cat. No. A26190). It is a fully outsourced bioinformatics solution for customers with little or no experience with bioinformatics, for help with data analysis.
The catalog number is A26190.
The Ion Universal Library Quantitation Kit is compatible with U-containing amplicons generated by the Ion 16S™ Metagenomics Kit whereas the Ion Library Quantitation Kit is not. For most other Ion libraries, use the Ion Library Quantitation Kit [exceptions: (1) RNA Seq libraries prepared using the Ion Total RNA-Seq Kit v2 and (2) amplicon libraries prepared following the Ion Amplicon Library Preparation (Fusion Method) User Guide, as these libraries have a truncated P1 (trP1) adapter sequence].
The components are listed below:
- TaqMan™ Fast Universal PCR Master Mix – 2 tubes (1.54 mL per tube)
- Ion Library TaqMan™ Quantitation Assay, 20X (250 µL)
- E. coli DH10B Ion Control Library – 2 tubes (25 µL per tube)
This may result in a lower observed library yield as determined by qPCR.
The Ion ReproSeq™ PGS (Preimplantation Genetic Screening) kits are bundles of four or five sub-kits which, when used in conjunction with the Ion PGM™ System, provide reagents and materials for whole genome amplification and sequencing to detect chromosomal aneuploidies, chromosome arm events (>48 Mb), and copy number variations starting from a single cell.
The Ion ReproSeq™ PGS workflow takes approximately 8-10 hours depending on the Ion Chip type in use. The library construction takes ~3.5 hours and the template preparation takes 2 hours. The run time for an Ion 314™ Chip takes 2.5 hours, an Ion 31™6 Chip takes 3.5 hours, and an Ion 318™ Chip takes 4.5 hours.
Six configurations of the Ion ReproSeq™ PGS Kit are available. The kits vary in the number of Ion SingleSeq kits provided, the maximum number of samples processed per kit, the type of Ion sequencing chip provided, and whether Ion sequencing chips are included.
Depending upon the configuration, there are 4 or 5 subkits in the Ion ReproSeq™ PGS kits:
- Ion SingleSeq Kit includes reagents to extract, amplify, and barcode genomic DNA.
- Ion PGM™ Template IA 500 Kit includes reagents for preparing template-positive Ion PGM™ Template IA Ion Sphere™ Particles (ISPs) for sequencing with the Ion PGM™ System.
- Ion PGM™ Hi-Q™ Sequencing Kit includes reagents and materials for the initialization and sequencing runs on the Ion PGM™ Sequencer.
- Ion PGM™ Hi-Q™ Wash 2 Bottle Kit
- Ion Chip Kit v2 - The Ion ReproSeq™ PGS kits are available with three different types of Ion Chips Kits v2: Ion 314™ Chip Kit v2, Ion 316™ Chip Kit v2, and Ion 318™ Chip Kit v2. The Ion Chips Kits v2 in the Ion ReproSeq™ PGS kits are barcoded (BC). The non-barcoded (non-BC) versions of the Ion Chip Kits v2 are also available and can only be purchased as standalone items.
The Ion ReproSeq™ PGS Kit workflow does not use the Ion OneTouch™ 2 System for template preparation. Template preparation is performed using Ion IA technology, whereby DNA is clonally amplified onto a bead surface through a non-emulsion, isothermal reaction.
Each barcode is intended for single use.
The Ion SingleSeq Kit and the Ion PGM™ Template IA 500 Kit are not available separately. The Ion PGM™ Hi-Q™ Sequencing Kit, Ion PGM™ Hi-Q™ Wash 2 Bottle Kit, and Ion Chip Kits v2 can be purchased as standalone items.
If only a subset of barcodes is needed, pierce the foil of the barcode adapter to collect the required amount of barcode adapter for the reaction. If you are using a subset, after piercing, seal the pierced wells with laboratory tape to minimize cross-contamination.
Do not exceed 4 freeze/thaw cycles.
1-10 cells are recommended for use with the Ion ReproSeq™ PGS Kit.
Purified gDNA can be used with the Ion ReproSeq™ PGS Kit and the recommended amount is 15-60 pg.
Sample libraries may be stored individually at -30 degrees C to -10 degrees C before proceeding to the next step. We do not recommend storing library pools prior to quantification.
Diluted library pools and non-diluted library pool stock solutions may be stored for 1 week at 4 degrees C. After 1 week, we recommend that you thaw the individual Ion SingleSeq libraries and repeat the pooling, purification, quantification, and dilution steps starting from the “Pool the libraries“ step in the protocol.
The template ISPs should be in the Ion PGM™ Template IA Wash Solution and can be stored at 4 degrees C for up to one week.
The enriched ISPs can be stored at 2 degrees C to 8 degrees C for up to 3 days.
The maximum number of Ion SingleSeq libraries that can be pooled and sequenced on a chip depends on the chip being used and can vary between 2-24 samples/run:
- Ion 314™ Chip Kit v2 (BC or non-BC) = 2 libraries per chip
- Ion 316™ Chip Kit v2 (BC or non-BC) = 16 libraries per chip
- Ion 318™ Chip Kit v2 (BC or non-BC) = 24 libraries per chip
Ion AmpliSeq™ Exome RDY Kits contain primers, library reagents, and chips for the rapid preparation and running of 8 exome libraries.
- Ion AmpliSeq™ Exome RDY Kit 1x8 (Cat. No. A27192) contains the Ion AmpliSeq™ Exome RDY Panel 1x8 (dried down oligo pools/primers in one 96-well plate with all 8 rows filled (1x8), for ultra-high multiplex PCR enrichment of the exonic regions of the genome)
- Ion AmpliSeq™ Exome RDY Kit 4x2 (Cat. No. A27193) contains the Ion AmpliSeq™ Exome RDY Panel 4x2 (dried down oligo pools/primers in four 96-well plates, each with 2 rows (C and F) filled (4x2), for ultra-high multiplex PCR enrichment of the exonic regions of the genome)
Note: Both of these kits include a 4-pack of Ion PI™ Chip Kit v3 for sequencing on the Ion Proton™ Sequencer.
- Ion AmpliSeq™ Exome RDY S5 Kit 1x8 (Cat. No. A29854) and Ion AmpliSeq™ Exome RDY S5 Kit 4x2 (Cat. No. A29855) contain all of the same reagents as the Ion AmpliSeq™ Exome RDY Kit 1X8 and the Ion AmpliSeq™ Exome RDY Kit 4x2 respectively, but instead include the Ion 540™ Chip for use with the Ion S5™ or Ion S5™ XL Systems.
The Ion AmpliSeq™ Exome RDY panels cover >97% of CCDS (with 5 bp padding around exons), >19,000 coding genes, >198,000 coding exons, no UTRs, miRNAs, or ncRNAs.
The amplicon size ranges from 225-275 bp, the average insert size is ~ 202 bp.
The exome RDY primers are dried down into ready-to-use format. There are ~294,000 primer pairs across 12 primer pools.
Due to the number of amplicons and coverage needed, the Ion AmpliSeq™ Exome RDY panels have only been validated on the Ion Proton™ Sequencer, Ion S5™ and Ion S5™ XL Systems.
The Ion AmpliSeq™ Exome RDY and Ion AmpliSeq™ Exome RDY S5 kits are not recommended for use with FFPE samples, as the amplicon target sizes (225-275 bp) are larger than we recommend for degraded DNA input. Increasing the input amount will not help, as the issue will be that some or many of the amplicon targets may not be amplifiable due to the degraded (fragmented) nature of the FFPE-derived DNA. This could result in amplicon drop out and incomplete coverage of the intended targets. Further, there could be issues with reproducibility across samples of differing levels of degradation. For example, some samples may produce sufficient results, while others may completely fail or produce sub-par results.
If barcode balancing is a priority, qPCR is recommended for library quantification. If workflow and speed is a priority, we recommend using the Ion Library Equalizer™ Kit.
- Ion AmpliSeq™ Exome RDY Kit 1x8 (Cat. No. A27192) and Ion AmpliSeq™ Exome RDY S5 Kit 1x8 (Cat. No. A29854) have only one 96-well plate with all 8 rows filled (1x8). Each row of a plate has the 12 primer pools needed for 1 exome. So each plate has 8 exomes (1 exome per row).
- Ion AmpliSeq™ Exome RDY Kit 4x2 (Cat. No. A27193) and Ion AmpliSeq™ Exome RDY S5 Kit 4x2 (Cat. No. A29855) come with 4 x 96-well plates, each with 2 rows (Rows C and F) filled (4x2). So there are 2 exomes per plate. The other wells on the plate are empty.
The plates are good for three rounds of target amplification. You can actually cycle the plates with the dried down wells for 3 rounds of cycling, and the dried down wells are still okay, so there is no need to cut the plate.
However, you may want to consider purchasing the Ion AmpliSeq™ Exome RDY Kit 4 X2 or Ion AmpliSeq™ Exome RDY S5 Kit 4x2. These kits come with 4 x 96-well plates, each with 2 rows (Rows C and F) filled (4x2). So there are 2 exomes per plate.
Information for Ampliseq™ panels can be found at Ampliseq.com. Each panel has a detailed description, which includes the number of pools, number of amplicons per pool, as well as a “Download Panel Files” link, which will contain the BED files.
The Ion AmpliSeq™Library Kit PLUS is a component of the Ion AmpliSeq™ Exome RDY and Ion AmpliSeq™ Exome RDY S5 kits, and is not sold separately. It was specifically launched for these kits and has improved formulation components, more robust user variability, higher tolerance of pipetting error, and 1-2% better uniformity.
- In the Ion Torrent™ browser, go to the “plan” tab and select “templates”.
- Select “ampliseq.com” and “Ampliseq exome”.
- You will be asked to enter your username and password.
- Choose “exome Panel” and select “import selected”.
- Exome template should be created with all appropriates BED and analysis parameter files/json files.
With the normal protocol (assuming the DNA input is correct, i.e., 50-100 ng), you should get about 200-1000 pM without the library amplification. With the library amplification, you would get higher concentration (1-5 nM).
These are average yields, and sometimes this does vary. Keep in mind that the library would work even with lower yields. qPCR is more sensitive than BioAnalyzer for quantitation of yield, and gives a slightly different measurement as qPCR measures ampliplifiable DNA whereas BioAnalyzer just gives the total yield regardless of whether the DNA is good for amplification.
You can use the Human CEPH Genomic DNA Control from the Ion P1 Controls 200 Kit (Cat. No. 4488985)
DNA outside the region window will not interfere with template preparation or sequencing, but may lead to an overestimation of library concentration when using the Qubit™ 2.0 Fluorometer for library quantitation.
The total instrument run time is approximately 7 hours.
No hardware upgrades are required to run the Ion AmpliSeq™ Kit for Chef DL8 on the Ion Chef™ Instrument.
The Ion AmpliSeq™ Kit for Chef DL8 enables robust, automated Ion AmpliSeq™ library preparation from genomic DNA or RNA using the Ion Chef™ Instrument and 1- or 2-pool Ion AmpliSeq™ primer panels. The kit contains sufficient reagents and consumables to prepare up to 32 barcoded and equalized libraries that are ready for template preparation using either the Ion Chef™ System or the Ion OneTouch™ 2 System. Library preparation from RNA additionally requires the SuperScript™ VILO™ cDNA Synthesis Kit, ordered separately. The kit components are as follows:
- Ion AmpliSeq™ Chef Reagents DL8 (4 cartridges)
- Ion AmpliSeq™ Chef Solutions DL8 (4 cartridges)
- Ion AmpliSeq™ Chef Supplies DL8 (1 box with 4 inserts)
- Ion AmpliSeq™ Tip Cartridge DL8
- Frames PCR Foil Seal
- Enrichment Cartridge
- IonCode™ 0101-0132 in 96-well PCR Plates (dried) (1 set of 4 plates):
- IonCode™ 0101–0108 in 96 Well PCR Plate (red)
- IonCode™ 0109–0116 in 96 Well PCR Plate (yellow)
- IonCode™ 0117–0124 in 96 Well PCR Plate (green)
- IonCode™ 0125–0132 in 96 Well PCR Plate (blue)
Yes, fewer than 8 samples may be processed in a run, but keep in mind that a run consumes kit reagents for 8 samples regardless of the sample number.
Please add 10 ng of each of the 8 samples to each well in Column 1 (Rows A to H) of the PCR plate.
They are in the PCR plate, column 6 (Rows A to H).
Optional sample tracking on the Ion PGM™ Torrent Server allows you to automatically follow samples grouped in “Sample Sets” from library and template preparation to chip loading, sequencing, and data analysis.
Automated sample tracking from library preparation to template preparation and sequencing is supported only for libraries prepared from one Sample Set in one Ion AmpliSeq™ for Chef run. If libraries from multiple Ion AmpliSeq™ for Chef runs are super-pooled in a Library Sample Tube in an Ion Chef™ template run, you will need to enter sample information manually when setting up a Planned Run.
For libraries >200 bp we recommend using standard rather than FAST instrument run mode on the thermocycler.
The primer pools should be added to the Reagent Cartridge on the Ion Chef™ Instrument in positions A and B. Please see schematic on Page 20 of the User Guide. Tubes in Positions C and D can be left empty.
If you are processing 5 or fewer samples, we recommend that you quantify your output combined library by qPCR to ensure that an optimal concentration is used in templating reactions.
The Ion AmpliSeq™ Kit for Chef DL8 contains sufficient reagents and consumables for performing 4 Ion Chef™ runs, with up to 8 Ion AmpliSeq™ libraries prepared per Ion Chef™ run, so that would be 32 libraries in total. The libraries are normalized using the equalizer kit to 100 pM and pooled together into one tube to go back on the Ion Chef™ System for template preparation.
Yes, Ion AmpliSeq™ 5X panels require dilution to 2X before use on the Ion Chef™ Instrument. Add 180 μL Nuclease-free Water or Low TE to 120 μL 5X panel.
You can run 8 DNA or cDNA samples with custom or ready-to-use 1- or 2-pool Ion AmpliSeq™ primer panels. RNA samples must be manually reverse transcribed to cDNA with the SuperScript™ VILO™ cDNA Synthesis Kit (sold separately) prior to library construction on the Ion Chef™ Instrument.
After completion of a run, the Ion Chef™ Instrument holds the barcoded libraries in the tube loaded in Position D of the Reagents cartridge. Please see schematic on Page 20 of the User Guide. The tube in Position D will contain 700 μL of combined barcoded libraries. The libraries are at »100 pM (total combined library concentration) and are ready to use in template preparation. To avoid fluid loss due to evaporation, remove and cap the tube of combined barcoded libraries as soon as possible after run completion. After 24 hrs from the start of the run, the instrument chiller will stop actively cooling, and the sample will be held at 27 degrees C. Do not leave the tube in the instrument longer than 24 hrs after the start of the run. You can store unused portions of combined libraries at 4-8 degrees C for up to 1 month. For longer-term storage, store at –30 degrees C to –10 degrees C.
It takes 30 minutes to complete DNA preparation using the Ion AmpliSeq Direct FFPE Kit.
Yes, the Ion Ampliseq Direct FFPE Kit has been tested on large Ampliseq panels, with varying amounts of FFPE DNA input, resulting in high coverage uniformity.
The shelf life of the Ion AmpliSeq Direct FFPE Kit is 3 years from the date of manufacture.
Yes, we recommend using the Qubit Fluorometer as an optional method of quantitation.
We have data for storage at -20 degrees C for 3 months.
The Ion AmpliSeq Direct FFPE DNA workflow cannot be automated because the slide collection step is manual.
Yes, FFPE DNA derived from the Ion AmpliSeq Direct FFPE Kit kit can be used with both the manual and automated Ion AmpliSeq library processes.
No, transfer solution will continue to penetrate the entire FFPE slide.
No, the Ion Plus Fragment Library Kit (48 rxns) is only compatible with samples that have been mechanically sheared using a sonicator such as the Bioruptor UCD-200 or the Bioruptor NGS UCD-600 Sonication System.
No, only Ion AmpliSeq libraries can be made on the Ion Chef Instrument
The Ion Plus Fragment Library Kit, 48 rxns (Cat. No. A28950) provides reagents for preparing up to 96 libraries at 100 ng input, or up to 48 libraries at 1 μg input.
For Research Use Only. Not for use in diagnostic procedures.