- Genomic target deep sequencing
- Invasive tissue biopsies를 또하지 않도록
Near-perfect analytic specificity(>99.9999%)는 일반적인 암 유전변이를 위양성없이 확인하게 한다.
Digital sequencing 기술을 tissue-based sequecing 162 pairs 3-4기 암환자시료와 비교했더니 이 기술은 민감도 85%, 조직기반은 80%. 연속샘플 1000개의 임상 성공율은 99.8%.
Table of Contents
Digital sequencing employs pre-sequencing preparation of a digital library of individually tagged cfDNA molecules combined with post-sequencing bioinformatic reconstruction to eliminate nearly all false positives.
타겟 영역이 커짐에 따라 위양성율이 증가하므로, hotspot, hot exone 영역만 대상으로 함
이 방법은 300-400 gene tissue-based NGS panel에 비해 적지만, 실제 advanced cancer 환자의 3/4 이상을 탐지함
Digital sequencing methodology vs. traditional NGS #
Analytic specificity and sensitivity #
Assay robustness/interference from genomic DNA secondary to storage time and temperature variation #
Assessment of copy number variation (CNV) #
Observational data from 510 patient multicenter study and 1,000 consecutive samples in clinical practice #
Analysis of control samples from healthy persons #
Clinical validation of the cfDNA panel #
Materials and Methods #
Characteristics of the Guardant360 panel and clinical diagnostic assay #
유전자 목록은 FDA 승인된 맞춤 치료 가능 목록
Guardant360 target region was 78 kbps per sample, 8,000X (min average base coverage 3,000X and min Qscore 20)
2 10ml tubes of whole blood --> formaldehyde-free 보관하면 상온 7일 보관
cfDNA fragments are converted to digital sequence library.
Bioinformatics processing of cell-free DNA sequencing data and variant calling #
Digital Sequencing signal processing for removal of false positive errors.
각 strand를 별도로 태깅 - 분석시 매칭하여 오류 보정
- 150bp paire-end reads aligned to reference
- Trim_galore in cutadapt removes 3' adapters
- Using custom script, remove unaligned and low quality tails, adapters
- Ids all germline SNP, somatic SNV, remove errors
For CNV or amplification, z-score > 2.5758
Accuracy and Analytic Specificity for SNVs #
The primer sets (Primer ID Hs00326065_CE, Life Technologies) were designed to amplify a 509 bp region in HRAS at chr11:532242–535550. The forward primer (CTCTAGAGGAAGCAGGAGACAGG) binds at position chr11:532242–535550 and the reverse primer (CATCACTGGGTCATTAAGAGCAA) binds at position chr11:534081–534589.