Comparing NGS and NanoString platforms in peripheral blood mononuclear cell transcriptome profiling for advanced heart failure biomarker development
2LeukoLifeDx, Point Pleasant, NJ 08742, USA
3Nanostring Technologies, Seattle, WA 98109, USA
INTRODUCTION
MATERIALS & METHODS
Patients
Patient # | Age (yr) | HF intervention | Gender | 1-year survival |
---|---|---|---|---|
1 | 69 | HFC | Male | Yes |
2 | 67 | MCS | Male | No |
3 | 73 | HFC | Male | No |
4 | 23 | HTx | Male | Yes |
5 | 65 | HTx | Male | Yes |
6 | 45 | HFC | Male | Yes |
7 | 56 | HTx | Male | Yes |
8 | 48 | HTx | Male | Yes |
9 | 73 | HFC | Male | Yes |
10 | 71 | HTx | Male | Yes |
11 | 47 | HFC | Male | Yes |
12 | 46 | HFC | Male | Yes |
13 | 42 | HFC | Male | Yes |
14 | 57 | HTx | Male | No |
15 | 22 | HTx | Male | No |
16 | 21 | HFC | Female | Yes |
17 | 59 | HTx | Male | Yes |
18 | 57 | MCS | Male | Yes |
19 | 66 | HTx | Male | Yes |
20 | 60 | HFC | Male | Yes |
21 | 53 | MCS | Male | Yes |
22 | 50 | HTx | Male | Yes |
23 | 64 | HFC | Male | Yes |
24 | 21 | MCS | Male | Yes |
25 | 69 | HTx | Male | Yes |
Sample collection, processing and RNA purification
PBMC transcriptome next generation sequencing analysis
PBMC transcriptome NanoString nCounter analysis
CodeSet choice
Experimental design
Statistical analysis
RESULTS
Number of overlapping genes for comparison between NanoString and NGS data
Nanostring/NGS-intrasample correlation
Lane | Cartridge #1 | Cartridge #2 | Cartridge #3 |
---|---|---|---|
1 | Sample 2, 20 ng × 5 µl | Sample 14, 20 ng x 5 µl | Sample 1, 5 ng × 5 µl |
2 | Sample 3, 20 ng × 5 µl | Sample 15, 20 ng x 5 µl | Sample 1, 10 ng × 5 µl |
3 | Sample 4, 20 ng × 5 µl | Sample 16, 20 ng x 5 µl | Sample 1, 20 ng × 5 µl |
4 | Sample 5, 20 ng × 5 µl | Sample 17, 20 ng x 5 µl | Sample 1, 20 ng × 5 µl |
5 | Sample 6, 20 ng × 5 µl | Sample 18, 20 ng x 5 µl | Sample 1, 40 ng × 5 µl |
6 | Sample 7, 20 ng × 5 µl | Sample 19, 20 ng x 5 µl | Sample 1, 80 ng × 5 µl |
7 | Sample 8, 20 ng × 5 µl | Sample 20, 20 ng x 5 µl | Sample 2, 5 ng × 5 µl |
8 | Sample 9, 20 ng × 5 µl | Sample 21, 20 ng x 5 µl | Sample 2, 10 ng × 5 µl |
9 | Sample 10, 20 ng × 5 µl | Sample 22, 20 ng x 5 µl | Sample 2, 20 ng × 5 µl |
10 | Sample 11, 20 ng × 5 µl | Sample 23, 20 ng × 5 µl | Sample 2, 20 ng × 5 µl |
11 | Sample 12, 20 ng × 5 µl | Sample 24, 20 ng × 5 µl | Sample 2, 40 ng × 5 µl |
12 | Sample 13, 20 ng × 5 µl | Sample 25, 20 ng × 5 µl | Panel standard |
Nanostring/NGS-intersample correlation
Number of counts | Number of genes | NanoString mean counts | NGS mean TPM |
---|---|---|---|
0–25 | 128 | 16.9 | 1.25 |
26–100 | 152 | 51.1 | 7.87 |
> 100 | 454 | 2995.8 | 214.37 |
Assess assay sensitivity, reproducibility and robustness within the NanoString platform
Log NGS expression average | Average correlation | Expression level |
---|---|---|
< 1 | 0.21 | Low |
1–2 | 0.58 | Intermediate |
2–3 | 0.63 | High |
> 3 | 0.68 | High |
Gene order (average expression among 734 genes) | Log nanostring expression average | Average correlation | Expression level |
---|---|---|---|
1–100 | 0–3.1 | 0.07 | Low |
100–200 | 3.1–3.8 | 0.34 | Low |
200–300 | 3.8–4.8 | 0.59 | Intermediate |
300–400 | 4.8–5.7 | 0.64 | High |
400–500 | 5.7–6.4 | 0.67 | High |
500–600 | 6.4–7.2 | 0.75 | High |
Above 600 | > 7.2 | 0.70 | High |
DISCUSSION
Acknowledgments
References
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