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

2. 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.

3. 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]

4. Comparison DNA extraction kits
Qiagen & Promega

5. 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

6. 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

7. 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

8. 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 for further collaboration