NGS Library Preparation Support—Getting Started

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.

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).

The Ion Universal Library Quantitation Kit is compatible with U-containing amplicons generated by the Ion 16S™ Metagenomics Kit whereas the Ion Library TaqMan Quantitation Kit is not. For most other Ion libraries, use the Ion Library TaqMan 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.

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.

The recommended input DNA amount for the Precision ID DL8 Kit is 125 pg.

The Precision ID IonCode Barcode Adapters 1-96 Kit in 96-Well PCR Plate provides enough reagents for 960 reactions.

The Precision ID IonCode Barcode Adapters 1-96 Kit in 96-Well PCR Plate provides 10 reactions per barcode.

Up to 32 libraries can be multiplexed on an Ion 530 chip (Cat. No. A27764) with the Precision ID GlobalFiler NGS STR Panel v2.

The Precision ID GlobalFiler NGS STR Panel v2 provides enough reagents for 96 reactions.

The Ion AmpliSeq RNA ERCC Companion Panel is compatible with both manual and chef-ready versions of human and mouse Ion AmpliSeq transcriptome gene expression kits:

- Ion AmpliSeq Transcriptome Human Gene Expression Kit (Cat. No. A26325, A26326, or A26327)
- Ion AmpliSeq Transcriptome Mouse Gene Expression Kit (Cat. No. A36553, A36554, or A36555)
- Ion AmpliSeq Transcriptome Human Gene Expression Panel, Chef-Ready Kit (Cat. No. A31446)
- Ion AmpliSeq Transcriptome Mouse Gene Expression Panel, Chef-Ready Kit (Cat. No. A36412)

The Ion AmpliSeq RNA ERCC Companion Panel consists of 10 primer pairs targeting 10 transcripts from the ERCC RNA Spike-In Mix representing a 13log2 dynamic range. Chosen for general GC balance and amenability for linear amplification (TaqMan qPCR), the selected ERCC targets produce R2 > 0.9 under normal experimental conditions.

We recommend adding 1 µL of the Ion Ampliseq RNA ERCC Companion Panel per manual target amplification reaction and 8 µL per primer pool for an automated target amplification reaction on the Ion Chef Instrument.

We do not recommend using the Ion AmpliSeq RNA ERCC Companion Panel with FFPE samples or with any panels besides human and mouse Ion AmpliSeq transcriptome gene expression panels.

To view data from the Ion AmpliSeq RNA ERCC Companion Panel, Torrent Suite Software version 5.10 or later is required and Version 5.10.0.3 or later of the ERCC_Analysis plugin for Torrent Suite Software is required.

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.

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.

You can use the Human CEPH Genomic DNA Control from the Ion P1 Controls 200 Kit (Cat. No. 4488985)

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.

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.

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.

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.      

The Ion AmpliSeq Mouse TCR Beta SR Assay, RNA is compatible with a vast array of research sample types, including FFPE tissue, fresh-frozen (FF) tissue, sorted T cells, whole blood, periperal blood leukocytes (PBLs), and periperal blood mononuclear cells (PBMCs).

The sample RNA input can range from 25 ng to 1 µg.

The Ion AmpliSeq Mouse TCR Beta SR Assay, RNA is compatible with the Ion 530 Chip, Ion 540 Chip, and Ion 550 Chip.

The AgriSeq HTS Library Kit is specifically designed for high-throughput preparation of amplicon libraries for targeted genotyping-by-sequencing (GBS) applications in agrigenomics.

The AgriSeq HTS Library Kit is designed to run on the Ion Chef and Ion S5/S5XL systems.

You can interrogate up to 5,000 SNPs per sample with the AgriSeq HTS Library Kit.

The AgriSeq HTS Library Kit can yield up to 1.6 M genotypes per day.

The process takes 2 days from sample preparation through data analysis.

The AgriSeq workflow requires less than 4 hours of hands-on time, from sample preparation through data analysis.

10 ng of DNA per target amplification can be used with the Oncomine BRCA Research Assay. The DNA can come from multiple sources, including FFPE samples and whole blood.

The sample can be FFPE DNA or gDNA.

24 reactions can be performed with the Oncomine BRCA Research Assay, Manual Library Preparation.

For the Oncomine Immune Response Research Assay, the minimum input amount of RNA is 10 ng.

The Oncomine Immune Response Research Assay covers 395 genes.

The Oncomine Immune Response Research Assay content includes 395 genes across 36 functional annotation groups. These groups comprise genes associated with lymphocyte regulation, cytokine signaling, lymphocyte markers, checkpoint pathways, and tumor characterization.

No, it only includes reagents for creating the library. For a complete kit, please see the Oncomine Focus Assay, 318 Solution, Cat. No. A28548. It includes all the necessary reagents and consumables for library construction, template preparation with the Ion OneTouch 2 System, and sequencing on the Ion PGM System.

8 samples can be processed per IonCode plate. The kit contains 4 IonCode plates, hence, a total of 32 samples can be processed using the Oncomine Focus Assay, Chef-Ready Library kit.

For each target amplification reaction, use 300-30,000 copies of DNA (10 ng of mammalian gDNA) from normal or FFPE tissue.

Each reverse transcription reaction requires 10 ng of DNase-treated total RNA (≥1.43 ng/µL).

The Oncomine Breast cfDNA Research Assay v2 covers the following genes:
- ALT1
- ERBB3
- KRAS
- CCND1
- ESR1
- PIK3CA
- EGFR
- FBXW7
- SF3B1
- ERBB2
- FGFR1
- TP53

More information on the coverage and the difference between versions 1 and 2 of the assay can be found in the flyer: Liquid Biopsy Cell Free Research Assays.

The Oncomine Breast cfDNA Research Assay v2 has only been validated on the Ion 530 and 540 chips at this time.

The Oncomine Breast cfDNA Research Assay v2 has only been validated on the Ion 530 and 540 chips at this time.

You would need to use Torrent Suite Software version 5.2 or later in order to see Oncomine options.

For automated library preparation using the Ion Chef System, we offer the Oncomine Myeloid Research Assay - Chef-Ready (Cat. No. A36941).

The recommended controls are:
-AcroMetrix Oncology Hotspot Control, Cat. No. 969056 (https://www.thermofisher.com/order/catalog/product/969056)
-Seraseq Myeloid Fusion RNA Mix, Cat. No. 0710-0407 (https://www.seracare.com/Seraseq-Myeloid-Fusion-RNA-Mix-0710-0407/)
-Seraseq Myeloid Mutation DNA Mix, Cat. No. 0710-0408 (https://www.seracare.com/Seraseq-Myeloid-Mutation-DNA-Mix-0710-0408/)

The assay can be run on both the Ion PGM System and Ion S5/S5XL systems.
-PGM Plexy = 4 samples per Ion 318 Chip
-S5 Manual Library Prep Plexy = 12 samples per Ion 530 Chip
-S5 Chef Library Prep Plexy = 8 samples per Ion 530 Chip

The Oncomine Myeloid Research Assay GX is a comprehensive targeted next-generation sequencing (NGS) assay designed for sensitive detection of myeloid disorder-associated DNA mutations and fusion transcripts in blood and bone marrow samples.

We recommend using the MagMAX Cell-Free DNA Isolation Kit (Cat. No. A29319) or the MagMAX Cell-Free Total Nucleic Acid Isolation Kit (Cat. No. A36716).

1 library can be used on an Ion 530 chip, 4 libraries can be used on an Ion 540 chip, and 8 libraries can be used on an Ion 550 chip.

The assay has a 0.1% limit of detection (LOD) with 20 ng of cell-free DNA input.

We have validated 8 samples on an Ion 540 chip: 7 DNA samples plus one negative control PLUS 7 RNA samples plus one negative control per chip; RNA and DNA from the same sample; 16 barcodes on each chip (8 barcodes for DNA and 8 barcodes for RNA).

This panel is compatible with as little as 10 ng per pool input DNA or RNA, per library, from blood, bone marrow, fresh/frozen tissue or FFPE samples. Reverse transcription of each sample requires 12 ng of DNase-treated total RNA (≥2.5 ng/µL) for manual library preparation and 2 × 5 ng/primer pool plus overage for the RT reaction. Each manual DNA library target amplification reaction requires 10 ng (≥1.82 ng/µL) of mammalian gDNA from normal or FFPE tissue per pool (20 ng in total).

The recommended controls are:
-Acrometrix Oncology Hotspot Control, Cat. No. 969056 (https://www.thermofisher.com/order/catalog/product/969056)
-Seraseq FFPE Tumor Fusion RNA Reference Material v2, Cat. No. 0710-0129 (https://www.seracare.com/Seraseq-FFPE-Tumor-Fusion-RNA-Reference-Material-v2-0710-0129/)

This panel is compatible with as little as 10 ng per pool input DNA or RNA, per library, from blood, bone marrow, fresh/frozen tissue or FFPE samples. Chef-ready RNA library preparation requires 10 ng of DNase-treated total RNA (≥0.8 ng/µL) for each sample. Chef-ready DNA library preparation requires 10 ng (≥0.67 ng/µL) of gDNA in total.

A variety of research sample types including fresh-frozen tissue, whole blood, and sorted T cells are compatible with the Oncomine TCR Beta-LR Assay.

Up to 16 samples can be multiplexed on an Ion 530 chip, up to 4 samples can be multiplexed on an Ion 520 chip, and up to 4 samples can be multiplexed on an Ion 318 chip.

We recommend using T Cell Leukemia (Jurkat) Total RNA (Cat. No. AM7858) as a control. T cell Leukemia (Jurkat) Total RNA is derived from a cell line consisting of a single T cell clonotype. Running the Oncomine TCR Beta-LR Assay on Jurkat Total RNA should enable detection of a single clonotype.

Flexible RNA input amount of 10 ng (minimum) and up to 1 µg supports the identification of rare clones as well as abundant clones.

The input amount range is 1-50 ng and the recommended input amount is 20 ng. There is a high primer dimer peak when using <5 ng input. The higher input will shift the family size distribution (i.e., more reads will be needed to call out a variant). LOD (limit of detection) for SNV/small indels calls and fusion detection is more sensitive to the input amount whereas LOD for CNV calls is only slightly impacted by the input amount.

For the best results, we recommend using plasma fraction from whole blood with minimal-to-low level of hemolysis. To prevent hemolysis during blood collection, please follow guidelines provided here (http://blog.fisherbioservices.com/avoiding-hemolysis-in-blood-sample-collection-and-processing blog). The Oncomine Lung Cell-Free Total RNA (cfNA) Research Assay is compatible with FFPE samples.

Note: Plasma samples with minimal-to-mild hemolysis are recommended to achieve minimal SNV false positives.

K2 EDTA blood collection tubes are preferred and can be purchasd from any major lab supplier. You can also use Cell-Free DNA BCT tubes from Streck (Cat. No. 218962).

1 sample can be multiplexed on an Ion 318 chip, up to 6 samples can be multiplexed on an Ion 530 chip, and up to 24 samples can be multiplexed on an Ion 540 chip

The number of reads needed to detect single nucleotide variants (SNVs) for each library is ˜2.5 million. This determines hpw many samples can be multiplexed on a chip.

The starting material is formalin-fixed paraffin-embedded (FFPE) tumor samples. We recommend using 10 ng of DNA per primer pool. As there are 2 pools, total input is 20 ng of DNA. For each target amplification reaction, use 300-30,000 copies of DNA (10 ng of mammalian gDNA) from normal or FFPE tissue. Increasing the amount of DNA results in higher-quality libraries, especially when DNA quality or quantity is unknown. We recommend using 1 ng of gDNA(300 copies) only with high-quality, accurately quantified samples.

Calculation of the Mutation Load alone does not require as many reads as when performing variant calling. To calculate the Mutation Load alone, we recommend combining up to a maximum of 8 libraries on an Ion 540 Chip, or up to a maximum of 16 libraries on an Ion 550 Chip.

To perform variant calling along with calculation of the Mutation Load, we recommend multiplex sequencing of no more than 4 libraries on an Ion 540 Chip and no more than 6 libraries on an Ion 550 Chip, to achieve sufficient read depth for variant calls at a ≥5% allele frequency.

IonCode Barcode Adapters 1-384 Kit (Cat. No. A29751) is compatible with the Oncomine Tumor Mutation Load Assay, Chef-ready library preparation. The set includes 4 PCR plates containing a total of 384 unique barcodes:

- IonCode 0101-0196 in 96 Well PCR Plate (red)
- IonCode 0201-0296 in 96 Well PCR Plate (yellow)
- IonCode 0301-0396 in 96 Well PCR Plate (green)
- IonCode 0401-0496 in 96 Well PCR Plate (blue)

No, the Oncomine Tumor Mutation Load Assay is optimized for use with FFPE tissue. The in-house development of the assay specifically focused on three tissue types: melanoma, lung, and colon.

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 316 Chip takes 3.5 hours, and an Ion 318 Chip takes 4.5 hours.

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.

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.

Collibri kits contain Platinum SuperFi DNA Polymerase whose exceptionally strong proofreading activity helps ensure amplification of NGS libraries with supreme sequence accuracy, producing NGS error rates similar to PCR-free methods. The enzyme is a part of Collibri Library Amplification Master Mix, which is included in the kits with PCR option. Designed exclusively for NGS application, Collibri Library Amplification Master Mix is based on a proprietary reaction buffer that has been specially optimized for efficient and uniform amplification of NGS libraries regardless of GC content.

Note: Collibri DNA Library Prep kits are available with or without PCR library amplification.

PCR-free libraries of ≥4 nM concentration using Collibri PS DNA Library Preparation Kit can be prepared from as little as 500 ng input DNA.

PCR-free libraries of ≥4 nM concentration using Collibri ES DNA Library Preparation Kit can be prepared from as little as 100 ng input DNA.

Library size distribution and the absence of primer dimers and/or over-amplification products should be verified by an electrophoretic method (with Agilent 2100 Bioanalyzer instrument).

To achieve the highest-quality sequencing data, it is essential to create optimal cluster densities across the flow cell. The most accurate quantification methods to assess the quantity of adaptor-ligated molecules in the library are qPCR-based methods, and we recommend using the Collibri Library Quantification Kit. Quantification can be performed at different stages of the workflow, for example after the post-ligation cleanup or size-selection step (for PCR-free workflows), or after the post-amplification cleanup step (for amplified libraries).

Collibri PS DNA Library Prep Kit can be used for library construction from lower-quality DNA (such as FFPE samples). However, the yields of adaptor-ligated molecules are typically lower compared to the yields obtained with high-quality DNA.

Collibri ES DNA Library Prep Kit is not recommended for library construction from FFPE samples.

No. They are specifically designed for a product and cannot be used interchangeably.