We evaluated the results from patients that had their stool samples tested by whole genome enrichment NGS, and their nasopharyngeal swabs tested by RT-PCR for the presence of SARS-CoV-2. Of the 14 study participants, ten were symptomatic and tested positive for SARS-CoV-2 by RT-PCR, two asymptomatic individuals tested negative, and two other asymptomatic individuals did not undergo RT-PCR testing (Table 1). While all patients were asked to collect stools at baseline, the samples were collected from 2-38 days after RT-PCR testing. Patients 5, 7, and 13, who all tested PCR positive by nasopharyngeal swab, were treated by their primary care physicians. Patients 5 and 7, were treated with Hydroxychloroquine (HCQ), Azithromycin (Zpack), vitamin C (3000mg), vitamin D (3000IU), and zinc (50mg) for 10 days. Stools from patients 5 and 7 were collected 5 and 6 days respectively after therapy regimens were initiated, at which time, both patients reported symptom clearance. Similarly, after positive nasopharyngeal swab, patient 13 began treatment consisting of high dosages of vitamin C (up to 10,000mg), vitamin D (up to 3000IU), and zinc (up to 50mg). This patient’s stool was collected 4 days after beginning the 10-day treatment regimen. Again, dosage changes were not recorded; however, symptom clearance was noted before fecal collection.
The concordance of SARS-CoV-2 detection by enrichment NGS from stools among positive non-treated patients tested by RT-PCR nasopharyngeal analysis was 100% (7/7). Patient 8, who did not undergo nasopharyngeal analysis, tested positive for SARS-CoV-2 by NGS. The three patients (5, 7, 13) that received treatment and were asymptomatic prior to providing fecal samples, were tested negative by NGS. Asymptomatic patients 2 and 9, who tested negative by nasopharyngeal swab, were also negative by NGS, as was asymptomatic patient 14.
Table 1. Symptoms and SARS-CoV-2 testing results
Patient ID
|
Age
|
Sex
|
Baseline Symptoms
|
RT-PCR Date
|
RT-PCR Results
|
Symptoms at Stool Collection
|
Stool Collection Date
|
Fecal
NGS Results
|
Location by
US State
|
1
|
21
|
F
|
Fever, diarrhea, anosmia, O2 sat. <90%
|
03/15/20
|
+
|
Diarrhea
|
04/21/20
|
+
|
PA
|
3
|
23
|
F
|
Fever, diarrhea, O2 sat. <90%
|
03/23/20
|
+
|
Diarrhea
|
04/05/20
|
+
|
CA
|
4
|
58
|
M
|
Fever, diarrhea, anosmia, O2 sat. <90%
|
03/30/20
|
+
|
Same as at Baseline
|
04/05/20
|
+
|
AZ
|
6
|
26
|
F
|
Fever, anosmia, cough
|
04/02/20
|
+
|
Same as at Baseline
|
04/04/20
|
+
|
AZ
|
8
|
91
|
M
|
None
|
N/A
|
N/A
|
N/A
|
N/A
|
+
|
CA
|
10
|
53
|
M
|
Fever, cough, headache
|
04/29/20
|
+
|
Same as at Baseline
|
05/03/20
|
+
|
GA
|
11
|
23
|
M
|
Fever, cough, headache
|
04/29/20
|
+
|
Same as at Baseline
|
05/03/20
|
+
|
GA
|
12
|
23
|
M
|
Fever, cough, anosmia
|
04/29/20
|
+
|
Same as at Baseline
|
05/04/20
|
+
|
GA
|
5
|
26
|
F
|
Fever, cough, diarrhea
|
03/23/20
|
+
|
None
|
03/29/20
|
-
|
CA
|
7
|
45
|
F
|
Fever, cough
|
04/08/20
|
+
|
None
|
04/13/20
|
-
|
GA
|
13
|
20
|
M
|
Fever, cough
|
04/29/20
|
+
|
None
|
05/02/20
|
-
|
GA
|
2
|
66
|
M
|
None
|
04/21/20
|
-
|
None
|
04/30/20
|
-
|
CA
|
9
|
55
|
F
|
None
|
03/13/20
|
-
|
None
|
05/05/20
|
-
|
CA
|
14
|
45
|
M
|
None
|
N/A
|
N/A
|
None
|
05/08/20
|
-
|
CA
|
US State Abbreviations: PA, Pennsylvania; AZ, Arizona; GA, Georgia, CA, California
|
The enrichment NGS analysis of fecal samples collected from RT-PCR-positive patients demonstrated mean read depths of 1129.8x, 31.7x, 318.6x, 1924.6x, 1206.7x, 15.6x, 3075.3x, and 92.7x, against the SARS-CoV-2 genome in patients 1, 3, 4, 6, 8, 10, 11, and 12, respectively. The ATCC SARS-CoV-2 served as a positive control in this study, with 7856.3x mean read depth. The sequencing read mapping results are depicted in Figure 1 (read depths are denoted on the y-axis and specific genomic coordinates on the x-axis). At 10x minimum read depth, all but two samples achieved a 100% breadth of coverage of the Wuhan-Hu-1 SARS-CoV-2 reference genome (Table 2). In patients 3 and 10, only 35% and 59% of genome completeness were achieved, respectively.
The total number of SARS-CoV-2 mapped reads for patients 1, 3, 4, 6, 8, 10, 11, and 12 were 465645, 5984, 131582, 793603, 496852, 5929, 1270734, and 38256 respectively (ATCC SARS-CoV-2 positive control, 94693754 reads).
Table 2. Enrichment NGS metrics
Sample ID
|
Genome Coverage
(10x minimum read depth)
|
Number of Variants
|
Mapped Reads
|
Mean Depth
|
Patient 1
|
100%
|
11
|
465645
|
1129.8x
|
Patient 3
|
35%
|
11
|
5984
|
31.7x
|
Patient 4
|
100%
|
9
|
131582
|
318.6x
|
Patient 6
|
100%
|
10
|
793603
|
1924.6x
|
Patient 8
|
100%
|
10
|
496852
|
1206.7
|
Patient 10
|
59%
|
9
|
5929
|
15.6x
|
Patient 11
|
100%
|
10
|
1270734
|
3075.3x
|
Patient 12
|
100%
|
10
|
38256
|
92.7x
|
Following alignment and mapping of SARS-CoV-2, patient genomes were compared to the Wuhan-Hu-1 (MN90847.3) SARS-CoV-2 reference genome via One Codex’s bioinformatics pipeline to identify mutational variations (see Methods for details). This analysis identified nucleotide variants at positions nt241 (C → T) and nt23403 (A → G) across all positive patients, and variants at positions nt3037 (C → T) and nt25563 (G → T) in seven of the eight patients (Table 3). Interestingly, patients 8, 11, and 12 harbored the same set of variants, as did patients 4 and 6 (who were kindreds). Unique variants not identified in any of the other individuals were detected in patients 1, 3, 6, and 10, with patient 3 harboring the most distinct SARS-CoV-2 genome with eight unique variants, followed by patient 1 with seven. Collectively, there were thirty-three different mutations among the patients in which SARS-CoV-2 was detected by whole genome enrichment NGS. One limitation to the variant analysis was that the One Codex SARS-CoV2 pipeline does not identify putative amino acids changes. As a result, Table 3 only captures nucleotide level resolution, making it difficult to assess mutations effect and evolutionary relationships.
Table 3. SARS-CoV-2 genomic positions, variant changes, and frequencies across the positive patient cohort
Region (ORF)
|
Position
|
Variant
|
Patient 1
|
Patient 3
|
Patient 4
|
Patient 6
|
Patient 8
|
Patient 10
|
Patient 11
|
Patient 12
|
5’-UTR
|
241
|
C → T
|
100%
|
100%
|
100%
|
100%
|
100%
|
100%
|
100%
|
100%
|
1a
|
833
|
T → C
|
x
|
x
|
x
|
x
|
100%
|
x
|
100%
|
100%
|
1a
|
1059
|
C → T
|
x
|
x
|
100%
|
100%
|
99%
|
100%
|
100%
|
100%
|
1a
|
1758
|
C → T
|
x
|
x
|
100%
|
100%
|
x
|
x
|
x
|
x
|
1a
|
1973
|
C → T
|
x
|
x
|
x
|
87%
|
x
|
x
|
x
|
x
|
1a
|
3037
|
C → T
|
100%
|
x
|
100%
|
100%
|
100%
|
100%
|
100%
|
100%
|
1a
|
3078
|
C → T
|
x
|
89%
|
x
|
x
|
x
|
x
|
x
|
x
|
1a
|
4866
|
G → T
|
75%
|
x
|
x
|
x
|
x
|
x
|
x
|
x
|
1a
|
6720
|
C → T
|
93%
|
x
|
x
|
x
|
x
|
x
|
x
|
x
|
1a
|
8102
|
G → T
|
x
|
100%
|
x
|
x
|
x
|
x
|
x
|
x
|
1a
|
9401
|
T → C
|
x
|
x
|
x
|
x
|
x
|
64%
|
x
|
x
|
1a
|
9403
|
T → A
|
x
|
x
|
x
|
x
|
x
|
64%
|
x
|
x
|
1a
|
10870
|
G → T
|
x
|
x
|
100%
|
100%
|
x
|
x
|
x
|
x
|
1a
|
11123
|
G → A
|
x
|
x
|
100%
|
100%
|
x
|
x
|
x
|
x
|
1b
|
14408
|
C → T
|
100%
|
x
|
100%
|
100%
|
100%
|
x
|
100%
|
100%
|
1b
|
14877
|
C → T
|
x
|
100%
|
x
|
x
|
x
|
x
|
x
|
x
|
1b
|
16616
|
C → T
|
x
|
x
|
x
|
x
|
100%
|
x
|
100%
|
100%
|
1b
|
16848
|
C → T
|
100%
|
x
|
x
|
x
|
x
|
x
|
x
|
x
|
1b
|
18652
|
C → A
|
x
|
x
|
x
|
x
|
x
|
83%
|
x
|
x
|
1b
|
19989
|
T → G
|
x
|
100%
|
x
|
x
|
x
|
x
|
x
|
x
|
Spike
|
21576
|
T → G
|
x
|
83%
|
x
|
x
|
x
|
x
|
x
|
x
|
Spike
|
23264
|
G → A
|
x
|
75%
|
x
|
x
|
x
|
x
|
x
|
x
|
Spike
|
23403
|
A → G
|
100%
|
100%
|
100%
|
100%
|
100%
|
100%
|
100%
|
100%
|
Spike
|
23603
|
C → T
|
82%
|
x
|
x
|
x
|
x
|
x
|
x
|
x
|
3a
|
25563
|
G → T
|
x
|
100%
|
100%
|
100%
|
100%
|
100%
|
100%
|
100%
|
3a
|
25976
|
C → A
|
x
|
x
|
x
|
x
|
100%
|
x
|
100%
|
100%
|
8
|
27964
|
C → T
|
x
|
x
|
x
|
x
|
100%
|
x
|
100%
|
100%
|
Nucleoprotein
|
28881
|
G → A
|
100%
|
x
|
x
|
x
|
x
|
x
|
x
|
x
|
Nucleoprotein
|
28882
|
G → A
|
100%
|
x
|
x
|
x
|
x
|
x
|
x
|
x
|
Nucleoprotein
|
28883
|
G → C
|
100%
|
x
|
x
|
x
|
x
|
x
|
x
|
x
|
Nucleoprotein
|
28997
|
C → T
|
x
|
100%
|
x
|
x
|
x
|
x
|
x
|
x
|
Nucleoprotein
|
29019
|
A → T
|
x
|
100%
|
x
|
x
|
x
|
x
|
x
|
x
|
Nucleoprotein
|
29364
|
C → G
|
x
|
x
|
x
|
x
|
x
|
85%
|
x
|
x
|