Are there Ion Torrent fragment library kits available for use with my AB Library Builder System?
The currently available kits for the AB Library Builder System instrument includes:
- Ion Plus Library Kit for AB Library Builder System (Cat. No. 4477597)
- Ion Xpress Plus Library Kit for AB Library Builder System (Cat. No. 4477598).
Both kits require the use of the separately available Ion Plus Fragment Library Adapters Kit (Cat. No. 4476340) or the Ion Xpress Barcode Adapters kits and the Ion Plus and Ion Xpress Plus Protocol Card for AB Library Builder System (Cat. No. 4477687).
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
What is the difference between a DNA fragment library and a DNA amplicon library?
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 requires further shearing or fragmentation during library preparation. The library preparation protocol for long amplicons is similar to fragment libraries.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
The Bioanalyzer instrument trace of my fragment library shows a large peak at 70 bp or 90 bp. What can cause this? Is it okay to proceed to template preparation?
The ~70 bp or ~90 bp peak is likely standard or barcoded adapter dimers, respectively. Adapter dimers may form during the adapter ligation step and are usually removed during the size selection process. The adapter dimers will amplify on the Ion Torrent Ion Sphere particles during template preparation and decrease the overall throughput of usable sequencing reads; thus, we highly recommend removing the adapter dimers by performing an additional clean-up step prior to template preparation.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
I would like to perform Next-Generation sequencing but my final library yield is poor. What are some tips for improving my yield?
In addition to input RNA quality and accurate quantification, the clean-up and size selection steps are critical to generating a successful RNA-Seq library.
- Be sure to mix the nucleic acid binding beads well before dispensing, and follow the workflow and incubation times as closely as possible.
- Use fresh ethanol and pre-wet pipette tips prior to transferring ethanol, as the volume is critical for size selection.
- Remove residual ethanol before elution using a small-volume pipette. Do not over-dry or under-dry the beads.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
I want to prepare a DNA fragment library for Next-Generation sequencing. Which library preparation kit and protocol should I use?
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 (Pub. No. 4471989). Please also refer to the Decision Tree for DNA Sequencing on the Ion PGM System and the Decision Tree for the Ion Proton System for more information.
Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.
