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.

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The library preparation protocol for long amplicons is similar to fragment libraries. 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. 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.

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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.

A29854) and Ion AmpliSeq™ Exome RDY S5 Kit 4x2 (Cat. 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 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. • Ion AmpliSeq™ Exome RDY Kit 1x8 (Cat. A27192) and Ion AmpliSeq™ Exome RDY S5 Kit 1x8 (Cat.