Ion Xpress™ Plus Fragment Library Kit - FAQs

For long-term storage, libraries should be stored concentrated (dilutions recommended for template preparation are not intended for long term storage) in LoBind tubes in low TE in non-frost free freezer at -20 degrees C.

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No, the Ion Library TaqMan Quantitation Kit for qPCR library quantification is unable to differentiate amplifiable primer-dimers from library fragments. We recommend assessing the library size distribution, including checking for the presence of adapter dimers, using the Bioanalyzer instrument. Libraries containing adapter dimers will have sharp peaks at ~70 bp for non-barcoded libraries or ~90 bp for barcoded libraries.

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We recommend using the qPCR result, as quantification by qPCR is generally more accurate than quantification using the Bioanalyzer instrument.

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Currently, the Ion Library Equalizer Kit is validated and only recommended for use with up to 300-base read libraries.

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We highly recommend quantifying unamplified libraries using qPCR, as it specifically measures the amplifiable (or usable) library fragments present. Unamplified libraries will contain unadapted and improperly adapted fragments, which will be included in quantification data obtained if using the Bioanalyzer instrument. Library quantification on the Bioanalyzer instrument using the Agilent High Sensitivity DNA Kit is only recommended for amplified libraries, as the library amplification process selects for properly adapted library fragments.

The Bioanalyzer instrument can be used to assess the size distribution of both amplified and unamplified libraries. Please see the Ion Xpress Plus gDNA Fragment Library Preparation User Guide (Pub. No. 4471989) for detailed information. If using the Bioanalyzer instrument for quantification of unamplified libraries, please note that this method will generally underestimate the concentration of usable library fragments and a library input titration may be necessary to optimize template preparation.

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We recommend quantifying libraries using the Ion Library Quantitation Kit (Cat. No. 4468802) for quantitative real-time PCR (qPCR) or the Bioanalyzer instrument. Each method has advantages and disadvantages.

The qPCR method is the most accurate and is recommended for both unamplified and amplified libraries, as it specifically measures the amplifiable (or usable) library fragments present. Unamplified libraries will contain unadapted and improperly adapted fragments, which will be included in quantification data obtained if using the Bioanalyzer instrument. The main disadvantage to qPCR is that it does not provide any library size information.

Library quantification on the Bioanalyzer instrument using the Agilent High Sensitivity DNA Kit provides both library concentration and size distribution information. This is only recommended for amplified libraries, as the library amplification process selects for properly adapted library fragments. The library quantification information obtained using the Bioanalyzer instrument is generally not as accurate, as qPCR quantification.

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The final EDTA concentration must be ?0.1 mM in the DNA preparation for the Ion Shear Plus reaction (in 40 µL total reaction volume). If there is sufficient material, re-purify or ethanol precipitate the appropriate amount of the DNA preparation and resuspend in nuclease-free water or 10 mM Tris, pH 7.5-8 for this procedure.

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For samples with 50-100 ng input, we recommend enzymatic shearing with the Ion Shear Plus Reagents Kit. Samples with 1 µg input are compatible with both enzymatic and physical shearing methods (e.g., Bioruptor or Covaris sonication instruments). Physical fragmentation methods using sonication are generally more reproducible and reliable with higher input amounts (e.g., 1 µg).

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Begin by optimizing with only a portion (such as half or less) of the sample first and assessing the shearing or fragmentation profiles using the Agilent Bioanalyzer instrument and Agilent High Sensitivity DNA Kit to ensure the fragments are within the desired size range.

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The Ion Shear reaction is sensitive to EDTA concentration, ssDNA or RNA contamination, and operator handling. If you have sufficient material, it is best to repurify the sample and elute in water before proceeding. If you must keep the sample in the same buffer, optimize the shearing reaction time with only a portion (such as half or less) of the sample first. Shearing profiles can be assessed using the Agilent Bioanalyzer instrument and Agilent High Sensitivity DNA Kit.

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The Ion AmpliSeq Designer (www.ampliseq.com) supports custom designs for targets in the human and mouse genomes. There are two design option sizes 125-175 bp, recommended for degraded DNA samples, and 125-275 bp, recommended for standard DNA input. Ion Ampliseq technology is a simple, fast, and affordable targeted sequencing strategy based on ultrahigh-multiplex PCR. The library preparation is completed in as little as 3.5 hours using 10 ng of DNA per PCR. To learn more about Ion Ampliseq technology, please review the Ion Ampliseq FAQs.

For other genomes (non-human or non-mouse) or applications requiring longer reads (up to 400-base reads), we recommend designing your own amplicons.

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The 23 nt P1adapter sequence length (or trP1 sequence) is the minimum sequence required for compatibility with the Ion template preparation kits. The shorter sequence allows for longer inserts (less of the recommended library size is taken up by the adapter sequence) and is more cost efficient for fusion primer synthesis.

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The fusion primer approach is faster and does not require any additional kits; however, this approach requires the use of custom primers (supports both barcoded and non-barcoded) and may also require some PCR condition optimizations for target amplification. Libraries constructed using the fusion primer method and truncated P1 adapter sequence are not compatible with the Ion Library Quantitation Kit for qPCR library quantification or the Ion Library Equalizer Kit for library normalization.

The adapter ligation approach has a longer workflow and requires the Ion Plus Fragment Library Kit, but does not require any additional custom, fusion primers and is compatible with both the Ion Library Quantitation Kit and Ion Library Equalizer Kit.

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To maintain the highest amount of library complexity, it is recommended to use 1 µg of DNA input and omit the optional, final library amplification. In order to further reduce bias, fragment the DNA using a physical fragmentation, and use the Ion Library Quantitation Kit (Cat. No. 4468802) for qPCR quantification of the library. The Agilent 2100 BioAnalyzer can be used to assess the library size distribution, but it is not recommended for quantification of unamplified libraries.

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PCR primer design guidelines:
- Use standard guidelines to design PCR primers for your region of interest. For design assistance, use a web tool such as Primer3, available at http://frodo.wi.mit.edu/primer3.
- Design your primers so that any sequence variants of interest are located between the primers, so that those variants are not masked by the template-specific part of the primer sequences.
- When designing primers for short amplicons (<350 bp), design the primers so that the amplicons do not exceed the maximum insert size for the desired target read length of the library. If you would like to sequence through the entire insert, design the primers so that the amplicon does not exceed the target read length.

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Libraries with insert sizes that are longer than the recommended insert length may not amplify efficiently onto the Ion Sphere particles (ISPs) during template preparation. This will result in low quality ISPs that will likely have suboptimal sequencing results, including shorter than expected read lengths. We recommend either redesigning the target amplicon to be shorter or shearing the DNA to the recommended insert length.

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Ion PGM System
Target Read Length: 400 bases; Median Insert Size: ~410 bp; Median Library Size*: ~480 bp
Target Read Length: 300 bases; Median Insert Size: ~320 bp; Median Library Size*: ~390 bp
Target Read Length: 200 bases; Median Insert Size: ~260 bp; Median Library Size*: ~330 bp
Target Read Length: 100 bases; Median Insert Size: ~130 bp; Median Library Size*: ~200 bp
* The median library size includes the insert size plus the Ion A adapter or Ion Barcoded A adapter and the Ion P1 adapter sizes.

Ion Proton System
Target Read Length: 200 bases; Median Insert Size: ~200 bp; Median Library Size*: ~270 bp
Target Read Length: 150 bases; Median Insert Size: ~150 bp; Median Library Size*: ~220 bp
* The median library size includes the insert size plus the Ion A adapter or Ion Barcoded A adapter and the Ion P1 adapter sizes.

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Yes, the Ion library adapters A and P1 are compatible with both the Ion PGM and Proton systems. Currently, the Ion PGM System supports up to 400-base reads (or ~470 bp library sizes) and the Ion Proton System supports up to 200-base reads (or ~ 270 bp library sizes).

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High-quality RNA-free DNA is required. The quality of the input DNA has a significant impact on the quality of the resulting library. A number of commercially available kits are available for isolation of high molecular weight, RNA-free genomic DNA. If your purification did not contain an RNase I digest, please refer to Appendix C of the Ion Xpress Plus gDNA Fragment Library Preparation User Guide for information about assessing the integrity and size of your input DNA material and performing an optional RNase treatment procedure.

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The DNA input amount will depend on the amount of DNA that can be isolated from the sample of interest and the application. For example, in order to construct a library with the highest complexity (e.g., vertebrate genomic library), it is recommended to start with 1 µg of DNA input, fragment by sonication (to reduce bias), not to perform library amplification (to maximize complexity), and to quantify the library by qPCR. Lower input (50-100 ng) amounts are sufficient for lower complexity libraries, such as amplicon libraries, or when the starting material is limiting. Please note that a final library amplification may be required for DNA inputs ? 100 ng. Finally, keep in mind that certain library preparation protocols require high DNA input (e.g., TargetSeq, ChIP-Seq, and Mate-Paired libraries).

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For the preparation of short amplicon libraries, we recommend two options:

1. Ion Plus Fragment Library Kit (Cat. No. 4471252) and the Preparing Short Amplicons (<350 bp) Libraries Using the Ion Plus Fragment Library Kit (MAN0006846) User Bulletin. Briefly, this kit and protocol combination requires amplification of the short amplicon targets, end repair of the PCR products, followed by adapter ligation and library quality control/quantification.
2. Fusion primer method, as described in the Ion Amplicon Library Preparation (Fusion Method, Cat. No. 4468326) User Guide. Briefly, the targets of interest are amplified using fusion primers, or primers containing both the target and Ion adapter specific sequences, followed by PCR reaction clean up and library quality control/quantification.

For the preparation of long amplicon libraries we recommend the Ion Xpress Plus Fragment Library Kit (Cat. No. 4471269) and the Preparing Long Amplicons (>400 bp) Libraries Using the Ion Xpress Plus Fragment library kit (MAN0007044) User Bulletin. Briefly, this kit and protocol combination requires amplification of the amplicon targets, shearing of the long amplicons with the Ion Shear Plus Reagents, adapter ligation, size selection, and library quality control /quantification.

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The Ion A and P1 adapter sequences can be found in the Appendix of the Ion Xpress Plus gDNA Fragment Library Preparation User Guide (Pub. No. 4471989).

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The recommended median insert size is the size of the target DNA fragment without the Ion A and P1 adapter sequences. The recommended median library size is the size of the final library, or the target DNA fragment size + Ion A and P1 adapter sizes. The standard Ion A and P1 adapter sequences add ~70 bp to the target DNA fragment size, while the Barcoded A adapter and P1 adapter sequences add ~80 bp to the DNA fragment size.

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The Ion Plus Fragment Library Kit and Ion Xpress Plus Fragment Library Kit contain reagents sufficient for preparing ? 20 libraries at 100 ng DNA input and ? 10 libraries at 1 µg DNA input.

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No, the Ion Shear Plus Reagents Kit module is not currently available separately.

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The Ion Xpress Plus Fragment Library Kit (Cat. No. 4471269) and the Ion Plus Fragment Library Kit (Cat. No. 4471252) are both used for the construction of fragment DNA and amplicon DNA libraries. The Ion Xpress Plus Fragment Library Kit is the Ion Plus Fragment Library Kit plus the Ion Shear Plus Reagents Kit, which is used for enzymatic shearing of the input DNA for library preparation. The Ion Plus Fragment Library Kit should be selected if using physical fragmentation (e.g., Bioruptor or Covaris instruments) or if no DNA fragmentation is required (e.g., short amplicon input).

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The number of samples that can be multiplexed in a single sequencing run depends on the capacity of the chip, the size of the library, and the required coverage. A table of approximate capacities based on the size of the library and chip can be found in the Ion Ampliseq Preparation User Guide (https://tools.thermofisher.com/content/sfs/manuals/MAN0006735_AmpliSeq_DNA_RNA_LibPrep_UG.pdf) within the section “Strategies for combining Ion Ampliseq libraries.”

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