AgriSeq™ HTS Library Kit, 960 reactions - FAQs

Here are possible causes and recommended solutions:
-High molecular weight DNA may not have been removed during purification of the amplified library. No action is required.
-Inserts may have formed concatemers during the ligation step. We recommend reducing the nucleic acid input, requantifying using the Qubit 1X dsDNA HS Assay Kit, Qubit dsDNA HS Assay Kit, or the Quant-iT DNA Assay Kit, High Sensitivity and Qubit Fluorometer, and reducing the target amplification cycle number.
Note: High molecular weight DNA does not interfere with sequencing.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Here are possible causes and recommended solutions:
-The sample DNA may have been misquantified. We recommend requantifying using the Qubit 1X dsDNA HS Assay Kit, Qubit dsDNA HS Assay Kit, or the Quant-iT DNA Assay Kit, High Sensitivity and Qubit Fluorometer.
-More than 10 ng of sample DNA may have been used. We recommend adding the recommended amount of DNA (10 ng) or decreasing the number of target amplification cycles.
Note: Library yield is not indicative of quality.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

Here are possible causes and recommended solutions:
-The sample may have been misquantified. We recommend requantifying the sample DNA using the Qubit 1X dsDNA HS Assay Kit, Qubit dsDNA HS Assay Kit, or the Quant-iT DNA Assay Kit, High Sensitivity and Qubit Fluorometer.
-Residual ethanol from AMPure purification may have inhibited library amplification. We recommend carefully removing all drops of ethanol before library amplification, then centrifuging the plate, if needed. Repeat residual ethanol removal.
-The sample quality may have been low. We recommend adding more DNA or increasing target amplification cycles and ensuring that the input DNA is free of PCR inhibitors. Please contact Technical Support at techsupport@thermofisher.com for details.
-The PCR, digestion, or ligation reactions may have been inefficient. We recommend ensuring proper dispensing and mixing of viscous components at each step.
-The AMPure XP beads may have been overdried. We recommend not drying the AMPure XP beads for more than 1 min.
-The Normalization Beads may not have been washed. We recommend ensuring that the Normalization Beads are washed before use.
-The wrong Normalization Primers may have been used. We recommend using the Normalization Primers that are provided in the AgriSeq HTS Library Kit.
-Residual Normalization Wash may have been present after was. We recommend carefully removing all the Normalization Wash before elution.
Note: Library yield is not indicative of quality.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

When used in combination with other IonCode barcode adapters (Cat. Nos. A31173, A36546, and A44599) and the AgriSeq HTS Library Kit (Cat. Nos. A34143 and A34144), IonCode Barcode Adapters, 1153-1536 enables pooling up to 1,536 amplicon libraries to conduct multiplexed sequencing analysis.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

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

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

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

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

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

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

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

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

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

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

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

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.