**3. Results and discussion**

#### **3.1. Swinger RNAs in the human mitochondrial transcriptome**

**Table 1** summarises results from BLASTn analyses of the purified mitochondrial transcriptome [48] for the 23 swinger-transformed versions of the human mitogenome. In total 4120 reads aligned with the 23 swinger-transformed versions of the human mitogenome,


producing 841 contigs. The highest detected identity between a theoretical mitogenome swinger transformation and a read was 95.32% for transformation A → C → T → G → A, and the lowest identity was 88.94% for transformation A → T → C → G → A. The overall identity averaged at 92.86%. A previous swinger analysis of other transcriptomic data [39] found swinger transformations A ↔ G and C ↔ T least and most frequent, respectively. Here, swinger transformation A ↔ G remains the least frequent; C ↔ T is the second most

**Figure 1.** Total mitogenome coverages by swinger RNAs across the complete human mitogenome in previously analysed

Swinger RNAs in the Human Mitochondrial Transcriptome

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83

data [39] (y-axis) as a function of those obtained in current observations from purified mitochondrial lines.

Total mitogenome coverages by swinger RNAs for each transformation were plotted as a function of corresponding coverages from a previous analysis published in 2016 [39]. Coverages are positively correlated (Pearson correlation coefficient r = 0.669, one-tailed p = 0.0002, **Figure 1**). Coverages for the purified mitochondrial line transcriptomes are systematically

Swinger RNA coverages of each of the 17 mitogenomic regions (D-loop, 2 rRNAs, 13 CDs and the WANCY region) are in **Tables 2** and **3**, for analyses of current and previous Illumina data [39], respectively. Pearson correlation coefficients between swinger coverages were calculated considering (1) genes, i.e. for each gene across the 23 different transformations, and (2) for

greater than for those for previous analyses (supplementary data and **Table 1**) [39].

each swinger transformation, across the 17 different mitogenomic regions.

frequent, suggesting high reproducibility.

**3.2. Gene-level comparisons of swinger RNA coverages**

Current data are from purified mitochondrial lines, previous data are from complete human transcriptome, including cytosolic and mitochondrial transcriptomes. Columns 2–5: current analyses. Columns are (1) swinger transformation (includes lack of transformation), (2) aligning read numbers, (3) contig numbers, (4) mean identity between reads and transformed mitogenome and (5) total mitogenomic coverage by all swinger contigs. Columns 6–9 indicate corresponding data in the same order for the previous study.

**Table 1.** Total human mitogenome coverage by detected swinger RNAs from current (2018) and previous (2016) [39] analyses of two different datasets sequenced by Illumina.

**3. Results and discussion**

82 Mitochondrial DNA - New Insights

**3.1. Swinger RNAs in the human mitochondrial transcriptome**

**Table 1** summarises results from BLASTn analyses of the purified mitochondrial transcriptome [48] for the 23 swinger-transformed versions of the human mitogenome. In total 4120 reads aligned with the 23 swinger-transformed versions of the human mitogenome,

Regular 700 142 99.76 8479 400 69 100 7658 A ↔ C 181 42 93.94 1239 163 10 89.12 389 A ↔ G 448 5 92.20 184 6 2 87.26 97 A ↔ T 98 48 94.34 1376 186 17 90.63 593 C ↔ G 319 7 89.99 227 28 8 85.01 343 C ↔ T 338 51 90.59 1599 253 26 92.47 937 G ↔ T 435 35 92.43 1614 400 5 88.36 249 A ↔ C + G ↔ T 123 25 89.79 730 11 2 86.23 76 A ↔ G + C ↔ T 63 34 92.63 982 69 8 89.3 257 A ↔ T + C ↔ G 126 36 92.87 1112 97 11 94.8 395 A → C → G → A 80 25 93.81 **778** 21 12 90.59 439 A → C → T → A 160 52 92.99 1474 31 12 90.79 453 A → G → C → A 98 58 91.70 1729 43 17 93.13 573 A → G → T → A 98 39 91.29 1245 28 12 88.49 469 A → T → C → A 218 50 93.12 1578 363 20 92.13 810 A → T → G → A 84 29 94.08 873 12 5 90.21 190 C → G → T → C 122 43 94.15 1167 21 4 88.44 247 C → T → G → C 165 57 91.90 2058 126 17 86.42 791 A → C → G → T → A 140 45 92.33 1306 38 15 89.53 575 A → C → T → G → A 157 35 95.32 971 54 10 92.65 327 A → G → C → T → A 211 26 92.63 713 99 15 87.35 530 A → G → T → C → A 78 27 93.60 777 30 11 91.29 369 A → T → C → G → A 195 17 88.94 463 60 9 92.1 321 A → T → G → C → A 183 55 94.14 1874 115 16 94.78 601

Current data are from purified mitochondrial lines, previous data are from complete human transcriptome, including cytosolic and mitochondrial transcriptomes. Columns 2–5: current analyses. Columns are (1) swinger transformation (includes lack of transformation), (2) aligning read numbers, (3) contig numbers, (4) mean identity between reads and transformed mitogenome and (5) total mitogenomic coverage by all swinger contigs. Columns 6–9 indicate

**Table 1.** Total human mitogenome coverage by detected swinger RNAs from current (2018) and previous (2016) [39]

corresponding data in the same order for the previous study.

analyses of two different datasets sequenced by Illumina.

**Read Contigs Id Coverage Read Contigs Id Coverage**

**Figure 1.** Total mitogenome coverages by swinger RNAs across the complete human mitogenome in previously analysed data [39] (y-axis) as a function of those obtained in current observations from purified mitochondrial lines.

producing 841 contigs. The highest detected identity between a theoretical mitogenome swinger transformation and a read was 95.32% for transformation A → C → T → G → A, and the lowest identity was 88.94% for transformation A → T → C → G → A. The overall identity averaged at 92.86%. A previous swinger analysis of other transcriptomic data [39] found swinger transformations A ↔ G and C ↔ T least and most frequent, respectively. Here, swinger transformation A ↔ G remains the least frequent; C ↔ T is the second most frequent, suggesting high reproducibility.

Total mitogenome coverages by swinger RNAs for each transformation were plotted as a function of corresponding coverages from a previous analysis published in 2016 [39]. Coverages are positively correlated (Pearson correlation coefficient r = 0.669, one-tailed p = 0.0002, **Figure 1**). Coverages for the purified mitochondrial line transcriptomes are systematically greater than for those for previous analyses (supplementary data and **Table 1**) [39].

#### **3.2. Gene-level comparisons of swinger RNA coverages**

Swinger RNA coverages of each of the 17 mitogenomic regions (D-loop, 2 rRNAs, 13 CDs and the WANCY region) are in **Tables 2** and **3**, for analyses of current and previous Illumina data [39], respectively. Pearson correlation coefficients between swinger coverages were calculated considering (1) genes, i.e. for each gene across the 23 different transformations, and (2) for each swinger transformation, across the 17 different mitogenomic regions.


**Transformation**

A ↔ C A ↔ G A ↔ T C ↔ G C ↔ T G ↔ T A ↔ C + G ↔ T A ↔ G + C ↔ T A ↔ T + C ↔ G A → C → G → A A → C → T → A A → G → C → A A → G → T → A A → T → C → A A → T → G → A C → G → T → C C → T → G → C A → C → G → T → A 8.8 A → C → T → G → A 5.4 A → G → C → T → A 4.5 A → G → T → C → A 9.4

7.9

0.0 0.0 2.5 0.0 0.0

0.0

0.0

3.2

0.0

0.0

5.0

0.0

0.0

0.0

0.0

0.0

5.1

3.4

6.7

2.5

0.155

0.551

85

4.7

0.0

3.1

0.0

2.1

4.4

19.3

0.0

9.3

9.2

0.0

0.0

7.5

0.0

0.0

−0.077

0.768

4.1

0.0

0.0

8.7

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

4.2

12.8

0.0

0.765

0.000

2.2

1.4

6.6

7.4

0.0

0.0

24.2

0.0

0.0

0.0

0.0

4.4

6.0

10.1

0.0

0.571

0.017

http://dx.doi.org/10.5772/intechopen.80805

2.2

4.8

3.8

12.0

2.3

0.0

30.0

0.0

0.0

0.0

0.0

0.0

7.0

40.2

6.6

0.833

0.000

Swinger RNAs in the Human Mitochondrial Transcriptome

4.5

4.5

1.8

3.1

0.0

0.0

0.0

0.0

0.0

0.0

3.6

0.0

0.0

0.0

0.0

6.9

0.0

0.335

0.189

0.0

2.9

0.0

3.9

0.0

0.0

0.6

0.0

0.0

0.0

0.0

0.0

0.0

0.0

2.4

8.2

0.0

−0.263

0.308

5.0

0.0

5.6

0.0

7.5

0.0

7.3

0.0

33.8

0.0

5.1

10.1

0.7

5.9

5.7

21.1

0.0

0.67

0.003

11.1

0.0

2.2

2.2

3.9

0.0

0.0

0.0

19.3

0.0

0.0

0.0

0.0

5.3

2.5

14.3

0.0

0.825

0.000

10.7

3.0

0.0

0.0

6.5

0.0

0.0

0.0

17.4

46.9

0.0

0.0

9.8

2.1

6.8

7.8

0.0

0.32

0.210

8.2

2.8

0.0

0.0

7.7

0.0

1.9

0.0

0.0

0.0

0.0

0.0

0.0

4.9

1.7

9.3

1.1

0.117

0.654

6.2

0.0

0.0

0.0

14.3

0.0

1.9

6.7

0.0

0.0

0.0

11.3

0.0

0.0

1.5

8.4

0.0

0.045

0.863

0.0

0.0

4.9

4.3

9.8

0.0

2.1

0.0

17.9

0.0

0.0

0.0

0.0

2.4

2.5

0.0

0.0

0.646

0.005

3.1

3.0

0.0

0.0

2.8

0.0

0.0

0.0

0.0

16.9

0.0

9.0

9.4

0.0

1.9

6.7

0.0

0.212

0.414

0.0

0.0

0.0

0.0

0.0

0.0

2.7

0.0

0.0

0.0

0.0

9.8

0.0

0.0

0.0

0.0

0.0

−0.168

0.520

3.7

0.0

0.0

5.0

4.7

0.0

0.0

0.0

24.2

0.0

0.0

0.0

0.0

0.0

0.0

10.1

4.3

0.696

0.002

9.4

5.9

7.0

9.4

7.6

0.0

0.0

6.1

15.5

0.0

0.0

9.8

0.0

4.4

6.9

33.0

2.6

0.869

0.000

5.1

0.0

0.0

0.0

9.8

0.0

3.2

0.0

13.5

0.0

0.0

0.0

0.0

0.0

0.0

6.7

0.0

0.66

0.004

8.9

4.1

4.2

0.3

7.7

0.0

1.6

0.0

17.9

13.0

0.0

0.0

0.0

7.5

4.4

0.0

0.0

0.366

0.148

0.0

0.0

0.0

0.0

0.0

0.0

3.2

0.0

0.0

0.0

0.0

0.0

0.0

0.0

2.6

0.0

0.0

0.198

0.446

**1** 4.1

0.0

0.0

0.0

3.1

0.0

0.0

0.0

14.5

0.0

0.0

9.5

9.4

7.0

3.8

8.4

0.0

0.21

0.418

**2**

**3**

**4**

**5**

**6**

**7**

**8**

**9**

**10**

**11**

**12**

**13**

**14**

**15**

**16**

**17**

**r**

**P**



**Transformation**

A

A

A

C

C

G

A

A

A

A

A

A

A

A

A

C

C

A

A

A

A

A

A

→ T →

**Table 2.**

G

→

C

→

A

16.9

4.1

10.4 Percentage coverage of mitogenomic regions by swinger RNAs in this study.

8.7

17.8

13.0

6.1

10.4

26.6

6.8

0.0

0.0

0.0

4.4

17.1

39.2

17.7

→ T →

C

→

G

→

A

2.3

2.8

3.5

0.0

2.9

0.0

3.1

1.3

0.0

0.0

6.1

0.0

0.0

6.5

2.9

0.0

2.2

→

G

→ T →

C

→

A

2.9

0.0

3.7

2.9

7.5

2.6

5.0

0.0

0.0

11.2

5.1

0.0

9.8

2.8

7.5

13.9

4.6

→

G

→

C

→ T →

A

5.9

2.3

2.2

0.0

17.3

0.0

3.8

4.2

0.0

3.4

0.0

0.0

4.4

0.0

10.0

0.0

5.0

→

C

→ T →

G

→

A

10.4

3.0

5.3

0.0

5.1

15.6

1.7

8.0

0.0

4.0

7.5

8.4

0.0

1.5

11.6

16.4

9.6

→

C

→

G

→ T →

A

11.6

5.0

4.0

10.7

20.7

0.0

3.8

3.1

15.0

4.4

6.1

8.1

0.0

4.2

14.2

21.7

3.0

→ T →

G

→

C

14.4

0.6

4.7

10.3

12.4

0.0

8.2

14.2

41.1

7.6

3.7

0.0

7.1

24.5

13.8

46.5

14.8

→

G

→ T →

C

10.1

4.9

2.0

11.5

20.0

0.0

5.4

0.0

17.4

3.5

19.0

13.6

0.0

7.3

5.8

14.9

0.0

→ T →

G

→

A

7.0

2.7

3.3

2.5

8.0

0.0

7.0

6.4

15.0

8.4

0.0

9.2

0.0

3.1

7.5

2.1

6.8

→ T →

C

→

A

12.3

2.2

7.3

15.9

12.0

0.0

6.8

3.8

21.7

7.6

11.0

16.5

20.2

10.4

7.7

35.0

6.3

→

G

→ T →

A

10.8

2.7

3.6

5.9

12.7

0.0

3.4

6.7

28.5

13.1

11.7

7.5

0.0

9.2

10.8

18.9

2.8

→

G

→

C

→

A

12.6

9.6

6.7

8.7

19.3

0.0

7.4

0.0

41.1

10.4

14.0

7.5

15.5

13.9

10.8

27.8

4.6

→

C

→ T →

A

9.0

0.0

6.7

5.0

13.8

19.6

9.9

20.8

13.5

12.0

9.8

8.4

0.0

10.1

6.3

15.0

9.2

→

C

→

G

→

A

4.5

2.5

0.1

0.0

7.5

8.2

0.0

0.0

10.1

12.8

6.3

0.0

0.0

0.0

8.7

13.0

7.7

↔ T + C

↔

G

4.7

7.2

7.0

2.4

8.5

6.6

10.5

5.1

35.7

0.0

4.1

27.5

0.0

9.4

4.9

0.0

5.3

↔

G + C

↔ T

2.9

2.1

0.0

7.7

18.0

5.6

7.7

3.8

0.0

0.0

4.1

7.5

21.2

10.3

6.4

18.9

0.0

↔

C + G

↔ T

2.6

5.2

7.7

0.0

5.6

5.6

8.4

4.1

0.0

0.0

0.0

0.0

0.0

4.4

5.5

11.4

1.8

↔ T

↔ T

↔

G

0.0 13.9 20.9

0.0

10.6

3.8

13.3

0.0

0.0

0.0

33.3

13.4

0.0

0.0

0.0

13.2

18.9

46.9

9.6

2.5

9.9

11.3

8.8

0.0

0.1

1.5

28.5

13.5

6.8

9.5

0.0

5.4

17.9

41.5

11.4

0.0

0.0

0.0

6.4

4.6

1.9

0.0

11.6

0.0

0.0

8.4

6.7

0.0

0.0

7.2

0.0

↔ T

↔

G

0.0 9.4

2.8

8.3

3.2

15.9

11.5

1.7

3.1

22.2

0.0

7.7

7.5

8.4

20.4

10.6

20.2

2.5

84 Mitochondrial DNA - New Insights

0.0

0.0

0.0

0.0

0.0

2.4

0.0

0.0

0.0

0.0

9.0

0.0

0.0

2.7

0.0

6.0

↔

C

6.4

5.6

9.1

0.0

15.1

25.0

5.8

0.0

9.2

3.7

7.3

11.8

0.0

8.3

7.9

26.7

3.7

**D-loop**

**12S**

**16S**

**ND1**

**ND2**

**W-Y**

**CO1**

**CO2**

**ATP8**

**ATP6**

**CO3**

**ND3**

**ND4L**

**ND4**

**ND5**

**ND6**

**Cytb**


**Table 3.** Percentage coverage of mitogenomic regions by swinger RNAs from analyses of complete human transcriptomic data [39]. Most correlations are positive along both genewise (columns) and transformation-wise (rows) analyses when comparing **Tables 2** and **3** (last rows and last columns in **Table 3**). Focusing on transformations and comparing coverages across genes for each transformation, correlations are positive between **Tables 2** and **3** for 19 among 23 transformations (P = 0.00065, one-tailed sign test) with 10 correlations statistically significant at P < 0.05. Analyses at the gene level across transformations detect 14 among 17 positive correlations (P = 0.003, one-tailed sign

Across genes, at the transformation level, the strongest correlation was observed for transformation C ↔ T (**Figure 2**) with Pearson r = 0.869 and one-tailed P = 0.0000029. Across transformations, at the gene level, the strongest correlation was observed for the gene ND6 with Pearson r = 0.752 and one-tailed P = 0.000017 with highest coverage at C ↔ T transformation in both datasets (**Tables 2** and **3**). In order to test whether swinger coverage has more transformation than gene-specific effect, we calculated the combined P value using Fisher's method to combine P values, for the 23 swinger transformations and, separately, for the 17 mitogenome regions. The method sums -2xln(Pi) where i ranges from 1 to k (k = 23 for transformations and k = 17 for genome regions/genes). This yield combined P = 5.7 × 10−21 for transformations

**Figure 2.** Percentage coverage of C ↔ T-transformed swinger RNAs across genes in this study as a function of their coverages in previously analysed data [39]. ND6 has the highest coverage among all transformations. Data from **Tables** 

for genes. This indicates a 3× stronger effect of transformation

Swinger RNAs in the Human Mitochondrial Transcriptome

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87

test), and six correlations at the gene level have P < 0.05 (one-tailed).

and combined P = 1.93 × 10−<sup>7</sup>

**2** and **3**.

Most correlations are positive along both genewise (columns) and transformation-wise (rows) analyses when comparing **Tables 2** and **3** (last rows and last columns in **Table 3**). Focusing on transformations and comparing coverages across genes for each transformation, correlations are positive between **Tables 2** and **3** for 19 among 23 transformations (P = 0.00065, one-tailed sign test) with 10 correlations statistically significant at P < 0.05. Analyses at the gene level across transformations detect 14 among 17 positive correlations (P = 0.003, one-tailed sign test), and six correlations at the gene level have P < 0.05 (one-tailed).

Across genes, at the transformation level, the strongest correlation was observed for transformation C ↔ T (**Figure 2**) with Pearson r = 0.869 and one-tailed P = 0.0000029. Across transformations, at the gene level, the strongest correlation was observed for the gene ND6 with Pearson r = 0.752 and one-tailed P = 0.000017 with highest coverage at C ↔ T transformation in both datasets (**Tables 2** and **3**). In order to test whether swinger coverage has more transformation than gene-specific effect, we calculated the combined P value using Fisher's method to combine P values, for the 23 swinger transformations and, separately, for the 17 mitogenome regions. The method sums -2xln(Pi) where i ranges from 1 to k (k = 23 for transformations and k = 17 for genome regions/genes). This yield combined P = 5.7 × 10−21 for transformations and combined P = 1.93 × 10−<sup>7</sup> for genes. This indicates a 3× stronger effect of transformation

**Figure 2.** Percentage coverage of C ↔ T-transformed swinger RNAs across genes in this study as a function of their coverages in previously analysed data [39]. ND6 has the highest coverage among all transformations. Data from **Tables 2** and **3**.

**Transformation**

A

A

r P

→ T →

G

→

C

→

A

14.6 0.535 0.008 Columns 1–17 areas in **Table**

**Table 3.**

mitogenomic region/swinger transformations, comparing data in **Tables 2** and **3** by rows and columns, respectively.

Percentage coverage of mitogenomic regions by swinger RNAs from analyses of complete human transcriptomic data [39].

0.519 0.215

0.182

0.776

0.939

0.337 0.203

0.000

0.892

0.693 **2**. r and P (last two columns and last two rows, respectively) indicate linear Pearson correlation coefficients between coverages across

0.477

0.064

0.174

0.003

0.000

0.011

0.142 0.269

0.289

0.063

−0.017 0.21

−0.276

0.678 0.03

0.087

−0.156

0.393

0.294

0.586

0.752

0.522

0.0

0.0

0.0

9.1

0.0

2.7

0.0

16.9

0.0

0.0

0.0

0.0

2.9

8.6

13.3

0.0

0.767

0.000

86 Mitochondrial DNA - New Insights

→ T →

C

→

G

→

A 2.9

0.0

0.0

3.6

7.8

0.0

0.0

1.2

0.0

0.0

0.0

10.4

8.8

1.7

0.0

0.0

0.0

−0.255

0.323

**1**

**2**

**3**

**4**

**5**

**6**

**7**

**8**

**9**

**10**

**11**

**12**

**13**

**14**

**15**

**16**

**17**

**r**

**P**

across genes on coverage than a gene-specific effect across transformations. Hence, the most important unknown factor determines transformations. The genome region that is swingertranscribed is important but secondary.

[3] Faure E, Delaye L, Tribolo S, Levasseur A, Seligmann H, Barthélémy RM. Probable presence of an ubiquitous cryptic mitochondrial gene on the antisense strand of the cyto-

Swinger RNAs in the Human Mitochondrial Transcriptome

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