Anchored Multiplex PCR for Targeted Next Generation Sequencing

AMP Meeting, November 12-16 2013


Zongli Zheng*, Boryana Zhelyazkova, Divya Panditi, Hayley E. Robinson, A. John Iafrate*, Long Le*

Massachusetts General Hospital, Department of Pathology, Boston, MA, 02114, USA


Current clinical genotyping based on next generation seuqencing is mostly driven by targeted gene panels. Particularly in the field of cancer genotyping, the need for high sequencing depth to achieve both complete gene coverage and the analytical sensitivity required for detecting low frequency variants in heterogeneous specimens renders whole genome sequencing and whole exome sequencing impractical. In addition, there is an unmet demand for a rapid, focused, and economical variant confirmation sequencing method with the ability to detect single nucleotide variants, insertions/deletions, copy number changes, and rearrangements.

We have developed a novel multiplex polymerase chain reaction assay termed anchored multiplex PCR (AMP) which can detect these four types of mutations. The assay may be performed with low amounts of RNA or DNA in a one- or two-tube format using commercially available reagents, custom primers, and standard library preparation instrumentation in one working day and then sequenced by Ion Torrent or Illumina sequencing.

Targeting double-stranded cDNA generated from total RNA, we have developed a one-tube lung cancer panel to detect druggable rearrangements in ALK, ROS1, and RET for clinical testing without prior knowledge of the heterologous fusion partners. Targeting genomic DNA, we have designed a 96 loci assay with minimal optimization which showed 100% 100X and >99.9% 500X minimum fold coverage at the targeted bases. A similar genomic DNA based assay targeting 370 exons (52.3 kilobases) with 626 total amplicons in a two-tube format showed >97% 100X and >93% 500X minimum fold coverage at the targeted bases in an initial run without any optimization. With efficient primer design solutions and higher oligo synthesis capacity, our technique may scale many fold higher for rapid, facile target enrichment in clinical, discovery, and confirmation sequencing applications.

Fusion Transcript (Rearrangement) Detection using Targeted RNA Seq

AMP was applied to detect gene rearrangments by looking for unknown 5' and 3' fusions partners based on targeting the known respective 3' or 5' partners. Unmapped reads after bwa alignment were subjected to BLAT analysis for fusion detection. Shown on the left are a list of fusions that have been detected from a cohort of FFPE samples previously tested by FISH. A novel gene fusion involving MSN Exon 9 and ROS1 Exon 34 was discovered (top right). Another sample showed two in-frame splicing variants of a CD74-ROS1 gene fusion.

AMP Figure 1A Anchored MultiPlex PCR method
AMP Figure 1B Fusion Transcript Detect using Targeted RNA Seq

Table 1. Mapping and Targeting Metrics

For two representative 96-amplicon and 626-amplicon gDNA genotyping panels.

Sample Input DNA per reaction (ng) Total Reads Post Trimming Reads % Aligned Reads using % On -Target
BWA Blat Overall BWA Blat Overall
96-amplicon gDNA panel in 2 reactions, one MiSeq run for 23 different tumor FFPE samples
1 100 642,338 632,186 94.4 4.7 99.0 80.5 2.9 83.4
2 100 567,238 558,367 92.8 5.8 98.7 79.8 3.4 83.2
3 100 529,623 520,241 94.0 5.1 99.0 80.9 3.3 84.2
4 100 737,942 715,553 91.6 6.8 98.4 81.7 4.4 86.0
5 100 450,450 442,746 94.0 4.9 99.0 78.2 3.0 81.2
6 100 478,867 468,913 93.9 5.1 99.0 78.2 2.9 81.2
7 100 553,994 541,422 92.0 6.8 98.8 76.4 4.4 80.8
8 100 513,991 501,669 93.6 5.4 99.0 83.5 3.5 87.0
626-amplicon gDNA panel in 2 reactions, one Miseq run for 4 samples
9 Plat.Taq 250 2,610,297 2,608,220 97.2 2.3 99.5 86.8 1.0 87.8
10 Plat.Taq +TMAC 250 2,516,838 2,513,743 97.5 2.0 99.5 88.1 1.1 89.2
11 OneTaq 250 3,506,335 3,483,793 70.2 25.5 95.8 10.7 0.2 10.8
12 Phusion 250 2,670,374 2,666,972 94.0 5.1 99.1 65.8 0.7 66.6

Table 2. Sample gDNA Genotyping Results

Seven, previously genotyped, clincial formalin-fixed paraffin-embedded (FFPE) nucleic acid samples were processed with a 96-amplicon cancer AMP assay. Sequencing data were mapped using a hybrid BWA+Blat approach. dbSNP variants and those showing lower than 5% allele frequency were filtered away. The 7 samples correspond to samples 1-7 in Table 1.

Sample % Target Bases with Minimum Coverage Single Base Extension Genotyping and PCR Sizing Results AMP 96-Amplicon Assay Results
100x 500x
1 98.8 93.5 Wild Type None
2 99.2 93.8 Wild Type None
3 99.7 94.6 Wild Type None
4 99.9 92.1 KRAS c.34 G>A, 4% KRAS c.34 G>A, 1.04%; CTNNB1 c.17 G>A, 6.4%; CTNNB1 c.206 G>A, 6.8%; FGFR c.746 C>T, 6.3%
5 96.9 90.6 CTNNB1 c.98 C>T, 21%; EGFR Exon 19 15-bp del, 22% CTNBB1 c.98 C>T, 10.7%; EGFR Exon 19 15-bp del, 16.9%
6 98.1 91.3 ERBB2 Exon 20 3-bp ins, 29% ERBB2 Exon 20 3-bp ins, 21.3%
7 98.3 94.2 ERBB2 Exon 20 12-bp ins, 96% ERBB2 Exon 20 12-bp ins, 95%

Sample Exon Coverage for PTEN

Coverage data were displayed for PTEN from 96-amplicon AMP assay. Dark blue blocks on the x-axis represent the exons and their bp size below. Black pileup peaks represent reads mapped to the PTEN target while turquoise pileup peaks represent reads mapped to a psuedogene.

AMP Figure 2 Sample exon coverage for PTEN

AMP Scalability

An AMP assay targeting 370 exons was tested using 4 different polymerase conditions. Normalized coverage data relative to the mean coverage of condition 1 are shown. Greater than 97% of targeted bases showed 100X minimum coverage, and greater than 94% of targeted bases showed 500X minimum coverage using Platinum Taq Polymerase. The results were generated from a one-time manually mixed primer pool without any optimization.

AMP Figure 3 AMP Scalability

AMP Copy Number Detection Potential

(A) Consistent distribution of coverage across EGFR exons 18, 19, 20, and 21 for an EGFR amplified sample relative to 3 normal controls which showed less overall coverage. (B) EGFR amplification relative to the copy neutral BRAF gene also on chromosome 7. Notice the greater than 7 fold relative difference between EGFR:BRAF in the amplified sampled compared to the 3 controls. In both panels, numbers in parentheses represent the highest coverage for the tallest pileup peak. Sequencing data was mapped with CLC Bio Genomics Workbench v5.5.1.

AMP Figure 4 AMP copy number detection potential


  • Anchored multiplex PCR (AMP) represents an economical and efficient target enrichment strategy based on standard molecular biology reagents and instrumentation
  • AMP allows target enrichment for SNV, indel, copy number, and rearrangement detection
  • We are currently improving the technology to further test its scalability and also to make the assay even leaner

For Research Use Only. Not for use in diagnostic procedures. For Research Use Only. Not for use in diagnostic procedures.

*Drs. A. John Iafrate and Long Le contributed equally to this work. They are co-founders of ArcherDX which is commercializing this technology for NGS target enrichment. Dr. Zongli Zheng is a consultant for ArcherDX.

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For Research Use Only. Not for use in diagnostic procedures. For Research Use Only. Not for use in diagnostic procedures.