Quality Control Metrics for DNA Sequencing Byoung-Kee Yi, PhD Dae-Soon Son, PhD Samsung Medical Center HL7 Korea

Need for NGS Quality Control NGS for clinical application requires a precise quality control measure to ensure sufficient reliability. The current quality metrics for NGS as existing mean coverage or uniformity are insufficient because they do not provide quality metrics directed to all possible results for many assays that are performed.

Comparison FFPE manufacturing condition In saline & Formalin fixation time [Reference : Fresh] FFPE 제작 조건에 따라 결과가 다르다. (FFPE 제작조건은 병원마다, 국가마다 다양하다) 그림들은 fresh specimen을 이용하여 다양한 조건으로 FFPE를 제작하여 시퀀싱한 결과다. 조건에 따라 다른 결과를 낼 수 있음을 보여준다. [Saline : 0h – Formalin 24h] [Saline : 0h – Formalin 72h] [Saline : 24h – Formalin 24h] [Saline : 48h – Formalin 24h]

Comparison DNA extraction kits Qiagen & Promega

Scope Metadata (QC metrics) specification across “sequencing pipeline” Sample Preparation – Sample QC Library Preparation – Library QC Sequence Generation – Run QC Initial Data Processing – Data QC

Examples of QC metrics QC Item Unit Input Definition DNA purity (260/280 ratio) ratio Extracted genomic DNA Measured DNA purity by NanoDrop (Protein contamination) DNA purity (260/230 ratio) Measured DNA purity by NanoDrop (Organic material contamination) DNA concentration ng/ul Measured double strand DNA concentration by Qubit 2.0 Fluorometer Total DNA amount ug Total amount of obtained gDNA. This value was measured by Qubit 2.0 Fluorometer △Ct cycle Extracted FFPE DNA The parameter means FFPE DNA integrity. This ratio was measured by real-time PCR machine Median size Bp Median value of DNA size. This value was measured by 2200 TapeStation Qubit conc./Median size ratio None DNA concentration (Qubit) and median size The divided ratio of DNA concentration (Qubit) with median size (The ratio = The measured DNA concentration (ng) by Qubit 2.0 Fluorometer / Median size (bp)) DNA input ng Extracted gDNA Initial input DNA amount for library construction DNA concentration after pre-PCR Prepared library after pre-PCR This value is a concentration of library via the input gDNA after pre-PCR procedure. This value is determined after every step in the progress of the library preparation process as A-tailing, paired-end adaptor ligation, and amplification

Examples of QC metrics (cont.) QC Item Unit Input Definition DNA amount after pre-PCR ul Prepared library after pre-PCR The total amount of prepared library after pre-PCR DNA input for Hybridization ng The input amount of prepared library for hybridization with baits (or customized RNA probes). The recommended input amount is 750ng DNA concentration after post-PCR Prepared library after post-PCR The DNA concentration of finally prepared library Average libraries size bp Prepared library Average size of prepared libraries Average insert size Average size of insert in libraries Molarity after post-PCR nM Average libraries size and DNA concentration in prepared libraries The value is a molar concentration of prepare library GC content % bam, bed Calculation of GC ratio within target region Bases Q ≥ 30 FASTQ The conversion value of each read quality into Phred score PCR duplicates FASTQ, dedup bam The ratio of duplicates in FASTQ file. The bias event is generally occurred by PCR amplification procedure Mean Read depth Average of read depth in target region Uniformity (>50%) rate Rate of sites which are exceed 50% of mean depth TARGET BASES ≥ 100X The rate of the region over 100X in target region USABLE BASES ON BAIT The rate of reads depth covering bait region Number of total reads number FASTQ file The total number of reads in FASTQ file

Status of the project Approved as a PWI by ISO/TC215 Form 4 and an early draft will be presented in Liverpool, UK for NP ballot approval Scheduled Nov. 6-10, 2017 Our target is a (preferably) TS or IS Seek interested SMEs Will create qc4genseq@googlegroups.com for further collaboration