ASHG 2019: Comparison between Mutation Profiles of Paired Whole Blood and cfDNA Samples
Introduction
Liquid biopsies are increasingly becoming a tool of choice for researching cancer detection and monitoring. Cell-free DNA or cfDNA is simply small fragments of DNA circulating in bodily fluids. It is also known as circulating cell-free DNA (ccfDNA), circulating tumor DNA (ctDNA) and cell free-fetal DNA (cffDNA). Next-Gen Sequencing of cfDNA is coming into maturity as a non-invasive method to identify mutational profiles in many cancer types.
- Apoptotic or necrotic cell death results in near-complete digestion of native chromatin from normal cell, tumor or fetus.
- Each 160-175 bp DNA is wrapped ~1.67 times around one nucleosome. These protein-bound DNA fragments preferentially survive digestion and are released into the circulation, and can be recovered from peripheral blood plasma as cfDNA.
- Typical cfDNA peaks characterized by Agilent 2100 Bioanalyzer, with a main peak at 175 bp, second and third peaks at 350 and 525 bp.

Methods
Sample Preparation
Blood was collected from 3 donors in EDTA tubes. After the blood was delivered to the site 10ng of Horizon Multiplex I cfDNA Reference Standard Set was added to ½ of the blood collected from each donor. The whole blood was then centrifuged twice, for 2,000xg for 10 minutes and supernatant was moved to a fresh tube and for centrifuged a second time at 6,000xg for 30 minutes. The second supernatant was used for cfDNA extractions; and the lower phase was used for genomic DNA extractions. Both phases were then stored at -80°C. The plasma was thawed at 37°C. Half of plasma from each donor that did not have 10ng of Horizon Multiplex I cfDNA Reference Standard Set had 200ng of Horizon Multiplex I cfDNA Reference Standard Set added to it as a positive control. The plasma samples were then processed using Apostle MiniMax™. Due to the lag in separating the whole blood from the plasma the cfDNA was size-selected using SPRIselect for 200bp size. The blood was thawed at room temperature. The genomic DNA was extracted from blood using GenFind V3.
Sample Name |
Sample Type |
Treatment |
cfDNA_508plus |
Plasma |
10 ng of Horizon Multiplex I cfDNA Reference Standard Set added prior to plasma isolation |
cfDNA_509plus |
||
cfDNA_510plus |
||
Blood_508plus |
Lower Phase of Blood |
|
Blood_509plus |
||
Blood_510plus |
||
cfDNA_508C |
Plasma |
200 ng of Horizon Multiplex I cfDNA Reference Standard Set added after plasma isolation |
cfDNA_509C |
||
cfDNA_510C |
||
Blood_508C |
Lower Phase of Blood |
Nothing added |
Blood_509C |
||
Blood_510C |
Library Preparation
Following extraction of gDNA and cfDNA, the DNA was quantified using Quant-it Picogreen Assay for the gDNA and using Kappa hgQuant kit for the cfDNA. Library construction was done using 100 ng of DNA with the Swift Biosciences Accel-NGS 2S Hyb DNA Library Kit, following the library construction genes were enriched prior to sequencing using the Swift Biosciences Pan-Cancer Hyb Panel. The libraries were sequenced on a NextSeq 550.Sequencing Analysis
The sequencing was analyzed using Illumina Basespace; the reads were aligned to the genes enriched in the Swift Biosciences Pan-Cancer Hyb Panel by using the BWA enrichment application.Similar coverage of reads for cfDNA and gDNA
Sample Name+ |
Total Aligned Reads |
Percent Aligned Reads |
Targeted Aligned Reads |
Read Enrichment |
Padded Target Aligned Reads |
Padded Read Enrichment |
Blood_508C |
58,813,981 |
100% |
33333485 |
57% |
35026658 |
60% |
Blood_508plus |
22,512,598 |
100% |
13011447 |
58% |
13655481 |
61% |
Blood_509C |
17,747,434 |
100% |
11593225 |
65% |
12237570 |
69% |
Blood_509plus |
18,678,850 |
100% |
12752101 |
68% |
13427976 |
72% |
Blood_510C |
20,504,009 |
100% |
12370374 |
60% |
12973986 |
63% |
Blood_510plus |
16,742,634 |
100% |
11320236 |
68% |
11894055 |
71% |
cfDNA_508C |
26,162,565 |
100% |
17505630 |
67% |
17734578 |
68% |
cfDNA_508plus |
30,039,905 |
100% |
18265618 |
61% |
18567893 |
62% |
cfDNA_509C |
33,127,967 |
100% |
18915218 |
57% |
19157799 |
58% |
cfDNA_509plus |
8,382,099 |
100% |
5973231 |
71% |
6154497 |
73% |
cfDNA_510C |
32,309,659 |
100% |
19874851 |
62% |
20109025 |
62% |
cfDNA_510plus |
12,908,033 |
100% |
8773743 |
68% |
8907368 |
69% |
The table above shows the number of reads that were aligned and the number of reads that were aligned to the targeted genes. The samples containing gDNA had on average 25 million reads and the cfDNA had on average 24 million reads.
Sample Name |
Mean Region Coverage Depth |
Uniformity of Coverage (Pct > 0.2*mean) |
Target Coverage at 1X |
Target Coverage at 10X |
Target Coverage at 20X |
Target Coverage at 50X |
Blood_508C |
4489.2 |
99.70% |
100.00% |
100.00% |
100.00% |
100.00% |
Blood_508plus |
1739.2 |
90.40% |
100.00% |
100.00% |
100.00% |
99.90% |
Blood_509C |
1550.5 |
88.80% |
100.00% |
100.00% |
100.00% |
99.80% |
Blood_509plus |
1703.6 |
86.50% |
100.00% |
100.00% |
100.00% |
99.80% |
Blood_510C |
1651.4 |
91.10% |
100.00% |
100.00% |
100.00% |
99.80% |
Blood_510plus |
1513 |
87.40% |
100.00% |
100.00% |
100.00% |
99.80% |
cfDNA_508C |
2315.1 |
92.40% |
100.00% |
100.00% |
100.00% |
99.90% |
cfDNA_508plus |
2432.1 |
90.50% |
100.00% |
100.00% |
100.00% |
99.90% |
cfDNA_509C |
2523.4 |
99.70% |
100.00% |
100.00% |
100.00% |
100.00% |
cfDNA_509plus |
801.6 |
85.10% |
100.00% |
100.00% |
99.90% |
99.20% |
cfDNA_510C |
2634.5 |
93.70% |
100.00% |
100.00% |
100.00% |
100.00% |
cfDNA_510plus |
1166.7 |
88.90% |
100.00% |
100.00% |
99.90% |
99.70% |
The table above shows the mean coverage depth for each sample. The average coverage for both gDNA and cfDNA was ~91%. At least 99% of the genes had coverage at 50x. This indicates that the variants can be called with high confidence.
Variant differences are observed between cfDNA and gDNA
Reference Standard Gene |
Reference Standard Variant |
Reference Standard Coordinate |
Reference Standard mutation Type |
Samples where Reference Standard was found |
|
KRAS |
G12D |
25398284 |
C>T |
cfDNA_ 508C |
cfDNA_ 510C |
NRAS |
Q61K |
1.15E+08 |
G>T |
cfDNA_ 509C |
|
NRAS |
A59T |
1.15E+08 |
C>T |
cfDNA_ 509plus |
Sample Name |
SNVs |
SNV Het/Hom Ratio |
SNV Ts/Tv Ratio |
Indels |
Indel Het/Hom Ratio |
Blood_508C |
500 |
1.4 |
2.2 |
117 |
3 |
Blood_508plus |
500 |
1.4 |
2.2 |
181 |
6 |
Blood_509C |
588 |
2.4 |
2.5 |
201 |
9.6 |
Blood_509plus |
590 |
2.4 |
2.5 |
196 |
8.8 |
Blood_510C |
501 |
1.7 |
2.3 |
197 |
8 |
Blood_510plus |
502 |
1.8 |
2.2 |
183 |
7.3 |
cfDNA_508C |
955 |
8.3 |
2.6 |
426 |
25.6 |
cfDNA_508plus |
636 |
6.8 |
2.3 |
275 |
18.6 |
cfDNA_509C |
963 |
8.2 |
2.6 |
434 |
26.1 |
cfDNA_509plus |
717 |
8.3 |
2.5 |
304 |
32.8 |
cfDNA_510C |
944 |
8.3 |
2.6 |
455 |
31.5 |
cfDNA_510plus |
669 |
7.4 |
2.4 |
303 |
29.3 |
Because we were unable to detect all of the spiked cfDNA, we extended our analysis to all variants. The total number of SNVs and insertion and deletions can be seen in the table above. For all three of the donors the average total number of variants found in gDNA was 29 and for cfDNA was 55 (Figure 1). To further this analysis we examined the number of variants that were only found in cfDNA or only found in gDNA. The number of variants only found in gDNA was 10 fold less than the number of variants only found in cfDNA for all three donors (figure 2). Because the majority of the mutations were found in both, this could indicate that any variant found only in the cfDNA could be candidates for ctDNA analysis.
Conclusions
- Variants found only in cfDNA could be used as an initial screen for ctDNA analysis
- Apostle MiniMax™ and GenFind V3 can be used together to get a picture of germ line variants and cfDNA, potential ctDNA, variants
- These results show that a holistic view of a cancer subject can be gained by using one sample source, whole blood