Introducing TruSeq® Amplicon 3.0

Also co-authored by Eric Allen.

Recent advancements in the Illumina TruSeq Amplicon technology enable higher multiplexing of amplicons in a single assay. Combined with next-generation sequencing (NGS) from Illumina, NGS users can perform high throughput, high sensitivity genotyping experiments on Illumina Sequencers. The new TruSeq Amplicon 3.0 BaseSpace® Sequence Hub App introduces major improvements to support a variety of amplicon sequencing applications, including the recently launched TruSeq Genotype Ne product. TruSeq Genotype Ne is a fully customizable targeted genotyping by sequencing (GBS) solution. Key GBS features of TruSeq Amplicon 3.0 include:

  • Support for custom reference genomes, allowing a user to analyze amplicon data against their choice of FASTA file (previously uploaded to Sequence Hub).
  • Genotypes of Interest reporting, allowing a user to generate a tabular report of genotypes for each sample, which is analogous to genotyping array outputs.

Example usage of the Genotypes of Interest feature can be found in the example Project below. The Input VCF (variant call file) in this Project (found in the test_NA12878_GOI output files) can be used as a template and customized for use with other datasets.

We also upgraded the TruSeq Amplicon 3.0 analysis pipeline to include:

  • Improved alignment and variant calling. Note that we have removed the outdated GATK v1.6 and Starling germline variant callers, replacing them with the new Pisces germline variant caller (optimized for amplicon data).
  • Improved variant annotation engine
  • Improved QC metrics engine
  • Support for up to 384 samples in a single analysis run

The improved small variant calling using the Pisces germline caller is demonstrated below for two data sets:

  • Coriell sample NA12878 with an internally developed panel that contains several challenging indels run on NextSeq®.
  • Coriell sample NA12878 with the TruSight® Myeloid Panel and sequenced on MiSeq®.

NextSeq® 550: TruSeq Amplicon (Replicates of NA12878)

VCF BED SNV Recall SNV Precision Indel Recall Indel Precision
TSA 2-0 – GATK TSAVP A v3 98.91% 98.91% 91.30% 100.00%
TSA 2-0 – Starling TSAVP A v3 94.57% 100.00% 43.48% 86.96%
TSA 3-0 – Pisces TSAVP A v3 98.91% 100.00% 93.48% 100.00%

MiSeq™ v3: TruSight® Myeloid (Coriell & HorizonDx, Pool 1)

VCF BED SNV Recall SNV Precision Indel Recall Indel Precision
NA12878S6-TSAv2 TruSight Myeloid v1.0 97.22% 92.11% 25.00% 28.57%
NA12878S6-TSAv3 TruSight Myeloid v1.0 94.44% 100.00% 87.50% 100.00%
  • Variant calls were compared by analyzing the same sample replicate with the TruSeq Amplicon v2.0 and v3.0 apps, and then using the Variant Calling Assessment Tool v3.0 app for accuracy assessment vs Platinum Genomes gold reference variant calls.

We hope this update enables you to discover new insights. Stay tuned for more app announcements, and let us know if you have any questions.

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