NGS-based identification and tracing of microsatellite instability from minute amounts DNA using inter-Alu-PCR

Abstract

Sensitive detection of microsatellite instability (MSI) in tissue or liquid biopsies using next generation sequencing (NGS) has growing prognostic and predictive applications in cancer. However, the complexities of NGS make it cumbersome as compared to established multiplex-PCR detection of MSI. We present a new approach to detect MSI using inter-Alu-PCR followed by targeted NGS, that combines the practical advantages of multiplexed-PCR with the breadth of information provided by NGS. Inter-Alu-PCR employs poly-adenine repeats of variable length present in every Alu element and provides a massively-parallel, rapid approach to capture poly-A-rich genomic fractions within short 80–150bp amplicons generated from adjacent Alu-sequences. A custom-made software analysis tool, MSI-tracer, enables Alu-associated MSI detection from tissue biopsies or MSI-tracing at low-levels in circulating-DNA. MSI-associated indels at somatic-indel frequencies of 0.05–1.5% can be detected depending on the availability of matching normal tissue and the extent of instability. Due to the high Alu copy-number in human genomes, a single inter-Alu-PCR retrieves enough information for identification of MSI-associated-indels from ∼100 pg circulating-DNA, reducing current limits by ∼2-orders of magnitude and equivalent to circulating-DNA obtained from finger-sticks. The combined practical and informational advantages of inter-Alu-PCR make it a powerful tool for identifying tissue-MSI-status or tracing MSI-associated-indels in liquid biopsies.

Single duplex DNA sequencing with CODEC detects mutations with high sensitivity

“…Considering that CODEC WGS showed 290-fold lower residual indel frequencies than standard WGS (Extended Data Fig. 9 ) and that Illumina NGS is known to have an especially high indel error rate at DNA homopolymers, we explored if CODEC might improve MSI detection, which is an FDA-approved indication for immunotherapy (anti-PD1/PDL1) but challenging to detect at low tumor fraction such as from liquid biopsies 40 . Indeed, CODEC WGS on NA12878 showed lower frequencies of both insertions and deletions at mononucleotide microsatellites than standard WGS (Fig.…”

“…Plasma from DFCI protocol 05–055 (breast cancer, patient 95) 23 was derived from 10 to 20 cc whole blood in EDTA tubes. Snap-frozen colon adenocarcinoma stage II/III and paired normal tissue biopsies from treatment-naïve patient (MSI, patient 19) were obtained from the Massachusetts General Hospital Tumor Bank, and gDNA was extracted using the Blood and Tissue kit (QIAGEN, 69556) 40 . The sperm sample was obtained as part of an IRB-approved human subjects protocol at New York University Grossman School of Medicine 43 ( Supplementary Text ).…”

Microsatellite Instability Testing in Colorectal Patients with Lynch Syndrome: Lessons Learned from a Case Report and How to Avoid Such Pitfalls

“…As Alu makes up 11% of the genome, this technique extended the previously published sensitivity of ctDNA in MSI testing by up to two orders of magnitude. In addition, it allowed the identification of MSI from a proportion of 0.05-0.15% of cfDNA [38].…”

Microsatellite instability assessment is instrumental for Predictive, Preventive and Personalised Medicine: status quo and outlook

“…The sequencing coverage as low as 0.05 × is sufficient for accurately calling the MSI status [ 191 ] Inter-Alu-PCR TGS NA This tool combines MNR enrichment and a custom MSI-tracer algorithm to compare the MNR’s read length. It can distinguish the cfDNA from cancer patients with MSI and samples from healthy individuals [ 192 ] Explanatory notes: Prog. Language , programming language; Ref.…”