# Draft genome sequences of 142 Mycobacterium avium subsp. paratuberculosis strains isolated from naturally infected dairy cattle

Cyril Conde, Maxime Branger, Thierry Cochard, Marie-Noëlle Rossignol, Christine Fourichon, Arnaud Delafosse, Aurore Davergne, Alain Joly, David Ngwa-Mbot, Laurent Journaux, et al.

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Cyril Conde, Maxime Branger, Thierry Cochard, Marie-Noëlle Rossignol, Christine Fourichon, et al. Draft genome sequences of 142 Mycobacterium avium subsp. paratuberculosis strains isolated from naturally infected dairy cattle. Microbiology Resource Announcements, 2021, 10 (38), 5 p. 10.1128/mra.00697-21. hal-03388033

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# Draft Genome Sequences of 142 *Mycobacterium avium* subsp. *paratuberculosis* Strains Isolated from Naturally Infected Dairy Cattle

Cyril Conde,ª Maxime Branger,ª Thierry Cochard,ª Marie-Noëlle Rossignol,ᵇ Christine Fourichon,ᶜ Arnaud Delafosse,ᵈ
Aurore Davergne,ᵉ Alain Joly,ᵉ David Ngwa-Mbot,ᵉ Laurent Journaux,ᵇ Raphael Guatteo,ᶜ Laurent Schibler,ᵢ Franck Bietª

**ABSTRACT** *Mycobacterium avium* subsp. *paratuberculosis* is the etiological agent of Johne's disease in ruminants. Here, we report the annotated draft genome sequences of 142 *M. avium* subsp. *paratuberculosis* strains that were isolated from dairy cattle in France between 2014 and 2018. The genomes of these strains were sequenced using Illumina technology.

Mycobacterium avium subsp. paratuberculosis is an economically significant veterinary pathogen in cattle, sheep, and goat populations (1). M. avium subsp. paratuberculosis is a very clonal species with genomes characterized by a high homology (2). To study the diversity of M. avium subsp. paratuberculosis strains and their phylogeny, it is therefore essential to have their entire genome sequences available.

Therefore, this sequencing project was conducted to sequence a panel of 142 *M. avium* subsp. *paratuberculosis* strains isolated during a longitudinal study of infected bovine (3) to better define the genetic features and pangenomic characteristics of this veterinary pathogen.

The 142 *M. avium* subsp. *paratuberculosis* strains were isolated from fecal samples collected as part of a longitudinal study of naturally infected cattle (3). The *M. avium* subsp. *paratuberculosis* isolates were propagated on slopes of Middlebrook 7H11 agar, 0.5% (vol/vol) glycerol, 10% (vol/vol) Middlebrook oleic acid-albumin-dextrose-catalase (OADC) enrichment medium (Becton, Dickinson, Oxford, Oxfordshire, UK), and 2 µg/ml of mycobactin J (IdVet, France) at 37°C. The isolates were tested for the presence of the *M. avium* subsp. *paratubercu-losis*-specific sequence IS900 (4) and genotyped using the MLVA (4).

Mycobacteria were harvested in the early to mid-log phase of growth, and the cells were pelleted at room temperature for 10 min at 6,000 × g. The pellets were resuspended in 10 ml of TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA) and centrifuged again at 6,000 × g for 10 min. The semidried mycobacterial pellets were resuspended in 360 µl of lysis buffer T1 (Macherey-Nagel, Germany, NucleoSpin tissue kit), and the samples were transferred to a Lysing Matrix B tube (mpbio) containing 350 mg of 0.1 mm silica spheres. The samples were homogenized in a FastPrep FP120 cell disruptor at 3 × 20 s, speed 6, followed by centrifugation at 14,000 × g for 5 min. The supernatants were transferred to fresh sterile microcentrifuge tubes, 25 µl of proteinase K (20 mg/ml) (Macherey-Nagel) was added, and the samples

**Citation** Conde C, Branger M, Cochard T, Rossignol M-N, Fourichon C, Delafosse A, Davergne A, Joly A, Ngwa-Mbot D, Journaux L, Guatteo R, Schibler L, Biet F. 2021. Draft genome sequences of 142 *Mycobacterium avium* subsp. *paratuberculosis* strains isolated from naturally infected dairy cattle. *Microbiol Resour Announc* 10:e00697-21. https://doi.org/10.1128/MRA.00697-21.

**Editor** Irene L. G. Newton, Indiana University, Bloomington

**Copyright** © 2021 Conde et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

Address correspondence to Franck Biet, franck.biet@inrae.fr.

**Received** 23 July 2021

**Accepted** 28 August 2021

**Published** 23 September 2021

<footnote>ªINRAE, Université de Tours, ISP, Nouzilly, France</footnote>

<footnote>ᵇINRAE, GABI, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France</footnote>

<footnote>ᶜINRAE, UMR1300 Biologie, Epidémiologie et Analyse de Risque en Santé Animale; Centre Nantes-Angers, Nantes, France</footnote>

<footnote>ᵈGroupement de Défense Sanitaire Orne, Alençon, France</footnote>

<footnote>ᵉGDMA, Bois-Guillaume, France</footnote>

<footnote>ᵉGroupement de Défense Sanitaire Bretagne, Vannes, France</footnote>

<footnote>ᵉGDS France, Paris, France</footnote>

<footnote>ᵇIdele, Paris, France</footnote>

<footnote n="1">¹Alice, Paris, France</footnote>
<table><caption>TABLE 1 Sequencing metrics for the 142 *Mycobacterium avium* subsp. *paratuberculosis* strains analyzed</caption><thead><tr><th>Isolate</th><th>Genome size (bp)</th><th>No. of contigs</th><th>N<sub>50</sub> (bp)</th><th>Total no. of genes</th><th>Total no. of CDSs<sup>a</sup></th><th>G+C content (%)</th><th>ENA accession no.</th><th>Contig accession no.</th></tr></thead><tbody><tr><td>PICSAR1</td><td>4,788,264</td><td>169</td><td>66,775</td><td>4,572</td><td>4,509</td><td>69.32</td><td>ERS6092665</td><td>CAJSNF010000001</td></tr><tr><td>PICSAR10</td><td>4,806,270</td><td>342</td><td>33,849</td><td>4,633</td><td>4,570</td><td>69.31</td><td>ERS6092666</td><td>CAJSMR010000001</td></tr><tr><td>PICSAR100</td><td>4,790,864</td><td>141</td><td>77,512</td><td>4,585</td><td>4,522</td><td>69.32</td><td>ERS6377149</td><td>CAJSMZ010000001</td></tr><tr><td>PICSAR102</td><td>4,793,984</td><td>158</td><td>74,709</td><td>4,582</td><td>4,519</td><td>69.32</td><td>ERS6092667</td><td>CAJPZJ020000001</td></tr><tr><td>PICSAR103</td><td>4,791,349</td><td>169</td><td>68,061</td><td>4,580</td><td>4,517</td><td>69.32</td><td>ERS6092668</td><td>CAJSMU010000001</td></tr><tr><td>PICSAR104</td><td>4,785,660</td><td>212</td><td>51,002</td><td>4,583</td><td>4,521</td><td>69.31</td><td>ERS6092669</td><td>CAJSND010000001</td></tr><tr><td>PICSAR106</td><td>4,784,162</td><td>168</td><td>58,265</td><td>4,568</td><td>4,505</td><td>69.32</td><td>ERS6092670</td><td>CAJSNC010000001</td></tr><tr><td>PICSAR107</td><td>4,783,589</td><td>205</td><td>54,247</td><td>4,597</td><td>4,534</td><td>69.32</td><td>ERS6092671</td><td>CAJSMV010000001</td></tr><tr><td>PICSAR11</td><td>4,786,941</td><td>216</td><td>50,211</td><td>4,602</td><td>4,540</td><td>69.31</td><td>ERS6092672</td><td>CAJSMY010000001</td></tr><tr><td>PICSAR110</td><td>4,785,567</td><td>188</td><td>56,293</td><td>4,586</td><td>4,523</td><td>69.31</td><td>ERS6092673</td><td>CAJSMS010000001</td></tr><tr><td>PICSAR111</td><td>4,780,529</td><td>170</td><td>56,292</td><td>4,572</td><td>4,509</td><td>69.32</td><td>ERS6092674</td><td>CAJSNE010000001</td></tr><tr><td>PICSAR113</td><td>4,784,630</td><td>180</td><td>62,923</td><td>4,579</td><td>4,516</td><td>69.31</td><td>ERS6092676</td><td>CAJSNG010000001</td></tr><tr><td>PICSAR117</td><td>4,788,531</td><td>167</td><td>63,942</td><td>4,566</td><td>4,503</td><td>69.32</td><td>ERS6092678</td><td>CAJSMT010000001</td></tr><tr><td>PICSAR118</td><td>4,780,126</td><td>206</td><td>52,735</td><td>4,595</td><td>4,532</td><td>69.31</td><td>ERS6092679</td><td>CAJSNB010000001</td></tr><tr><td>PICSAR119</td><td>4,786,159</td><td>196</td><td>50,347</td><td>4,577</td><td>4,514</td><td>69.31</td><td>ERS6092680</td><td>CAJSMW010000001</td></tr><tr><td>PICSAR120</td><td>4,788,510</td><td>204</td><td>56,691</td><td>4,589</td><td>4,526</td><td>69.3</td><td>ERS6377150</td><td>CAJSMX010000001</td></tr><tr><td>PICSAR124B</td><td>4,787,722</td><td>136</td><td>86,432</td><td>4,570</td><td>4,507</td><td>69.32</td><td>ERS6377151</td><td>CAJSNA010000001</td></tr><tr><td>PICSAR124</td><td>4,792,645</td><td>158</td><td>71,665</td><td>4,573</td><td>4,510</td><td>69.32</td><td>ERS6092682</td><td>CAJSNI010000001</td></tr><tr><td>PICSAR126</td><td>4,796,308</td><td>164</td><td>86,432</td><td>4,561</td><td>4,498</td><td>69.33</td><td>ERS6092684</td><td>CAJSNH010000001</td></tr><tr><td>PICSAR13</td><td>4,789,268</td><td>146</td><td>80,438</td><td>4,569</td><td>4,506</td><td>69.32</td><td>ERS6092687</td><td>CAJSNJ010000001</td></tr><tr><td>PICSAR131</td><td>4,790,689</td><td>128</td><td>92,490</td><td>4,561</td><td>4,498</td><td>69.32</td><td>ERS6377152</td><td>CAJSNU010000001</td></tr><tr><td>PICSAR132</td><td>4,787,497</td><td>177</td><td>63,521</td><td>4,583</td><td>4,520</td><td>69.32</td><td>ERS6092689</td><td>CAJSNQ010000001</td></tr><tr><td>PICSAR135</td><td>4,794,273</td><td>153</td><td>91,217</td><td>4,583</td><td>4,520</td><td>69.33</td><td>ERS6092690</td><td>CAJSNR010000001</td></tr><tr><td>PICSAR136</td><td>4,788,400</td><td>184</td><td>59,721</td><td>4,586</td><td>4,523</td><td>69.32</td><td>ERS6092691</td><td>CAJVAR010000001</td></tr><tr><td>PICSAR14</td><td>4,787,453</td><td>243</td><td>41,260</td><td>4,609</td><td>4,546</td><td>69.31</td><td>ERS6092693</td><td>CAJSNP010000001</td></tr><tr><td>PICSAR140</td><td>4,789,215</td><td>126</td><td>92,229</td><td>4,563</td><td>4,500</td><td>69.33</td><td>ERS6092694</td><td>CAJSNW010000001</td></tr><tr><td>PICSAR141</td><td>4,786,839</td><td>168</td><td>71,205</td><td>4,574</td><td>4,511</td><td>69.31</td><td>ERS6092695</td><td>CAJSNX010000001</td></tr><tr><td>PICSAR142</td><td>4,787,534</td><td>161</td><td>64,094</td><td>4,581</td><td>4,518</td><td>69.32</td><td>ERS6092696</td><td>CAJSNS010000001</td></tr><tr><td>PICSAR143</td><td>4,779,428</td><td>192</td><td>55,835</td><td>4,570</td><td>4,507</td><td>69.32</td><td>ERS6092697</td><td>CAJSNT010000001</td></tr><tr><td>PICSAR145</td><td>4,785,805</td><td>163</td><td>64,178</td><td>4,575</td><td>4,512</td><td>69.32</td><td>ERS6092699</td><td>CAJSNV010000001</td></tr><tr><td>PICSAR147</td><td>4,789,937</td><td>175</td><td>68,132</td><td>4,576</td><td>4,513</td><td>69.32</td><td>ERS6092701</td><td>CAJSNO010000001</td></tr><tr><td>PICSAR148</td><td>4,788,670</td><td>141</td><td>79,766</td><td>4,561</td><td>4,498</td><td>69.32</td><td>ERS6092702</td><td>CAJSNM010000001</td></tr><tr><td>PICSAR15</td><td>4,780,402</td><td>214</td><td>49,347</td><td>4,581</td><td>4,518</td><td>69.31</td><td>ERS6092704</td><td>CAJSNY010000001</td></tr><tr><td>PICSAR154</td><td>4,789,709</td><td>162</td><td>70,010</td><td>4,586</td><td>4,523</td><td>69.32</td><td>ERS6092706</td><td>CAJSNL010000001</td></tr><tr><td>PICSAR157</td><td>4,787,040</td><td>166</td><td>62,990</td><td>4,573</td><td>4,510</td><td>69.32</td><td>ERS6092707</td><td>CAJSNN010000001</td></tr><tr><td>PICSAR16</td><td>4,789,808</td><td>170</td><td>64,202</td><td>4,578</td><td>4,515</td><td>69.32</td><td>ERS6092708</td><td>CAJSNK010000001</td></tr><tr><td>PICSAR160</td><td>4,787,812</td><td>173</td><td>66,624</td><td>4,566</td><td>4,503</td><td>69.32</td><td>ERS6092709</td><td>CAJSOD010000001</td></tr><tr><td>PICSAR162</td><td>4,785,890</td><td>195</td><td>49,541</td><td>4,584</td><td>4,521</td><td>69.31</td><td>ERS6377153</td><td>CAJSOA010000001</td></tr><tr><td>PICSAR164</td><td>4,769,542</td><td>325</td><td>30,069</td><td>4,608</td><td>4,545</td><td>69.29</td><td>ERS6092710</td><td>CAJSOC010000001</td></tr><tr><td>PICSAR168B</td><td>4,790,339</td><td>151</td><td>86,432</td><td>4,561</td><td>4,498</td><td>69.33</td><td>ERS6377154</td><td>CAJSOB010000001</td></tr><tr><td>PICSAR168</td><td>4,790,212</td><td>149</td><td>74,685</td><td>4,563</td><td>4,500</td><td>69.32</td><td>ERS6092711</td><td>CAJSNZ010000001</td></tr><tr><td>PICSAR172</td><td>4,788,168</td><td>144</td><td>74,172</td><td>4,569</td><td>4,506</td><td>69.32</td><td>ERS6092713</td><td>CAJSOE010000001</td></tr><tr><td>PICSAR178</td><td>4,773,707</td><td>183</td><td>58,645</td><td>4,571</td><td>4,508</td><td>69.32</td><td>ERS6092714</td><td>CAJSOH010000001</td></tr><tr><td>PICSAR179</td><td>4,788,184</td><td>192</td><td>59,069</td><td>4,587</td><td>4,524</td><td>69.32</td><td>ERS6092715</td><td>CAJSOK010000001</td></tr><tr><td>PICSAR18</td><td>4,783,336</td><td>218</td><td>47,690</td><td>4,594</td><td>4,532</td><td>69.31</td><td>ERS6092716</td><td>CAJSOM010000001</td></tr><tr><td>PICSAR180</td><td>4,790,473</td><td>144</td><td>79,784</td><td>4,568</td><td>4,505</td><td>69.32</td><td>ERS6092717</td><td>CAJSOG010000001</td></tr><tr><td>PICSAR181</td><td>4,783,299</td><td>213</td><td>48,895</td><td>4,583</td><td>4,520</td><td>69.31</td><td>ERS6092718</td><td>CAJSOI010000001</td></tr><tr><td>PICSAR182</td><td>4,786,048</td><td>199</td><td>53,495</td><td>4,576</td><td>4,513</td><td>69.32</td><td>ERS6092719</td><td>CAJSOF010000001</td></tr><tr><td>PICSAR184</td><td>4,789,623</td><td>168</td><td>66,614</td><td>4,575</td><td>4,512</td><td>69.32</td><td>ERS6092720</td><td>CAJSOJ010000001</td></tr><tr><td>PICSAR189</td><td>4,788,806</td><td>179</td><td>69,522</td><td>4,565</td><td>4,502</td><td>69.32</td><td>ERS6092721</td><td>CAJSOL010000001</td></tr><tr><td>PICSAR19</td><td>4,789,870</td><td>163</td><td>64,491</td><td>4,569</td><td>4,506</td><td>69.32</td><td>ERS6092722</td><td>CAJSOQ010000001</td></tr><tr><td>PICSAR191</td><td>4,789,697</td><td>173</td><td>67,710</td><td>4,564</td><td>4,501</td><td>69.32</td><td>ERS6092723</td><td>CAJSOR010000001</td></tr><tr><td>PICSAR192</td><td>4,788,235</td><td>193</td><td>58,604</td><td>4,595</td><td>4,532</td><td>69.31</td><td>ERS6092724</td><td>CAJSOU010000001</td></tr><tr><td>PICSAR196</td><td>4,791,434</td><td>161</td><td>71,932</td><td>4,570</td><td>4,507</td><td>69.32</td><td>ERS6092725</td><td>CAJSOS010000001</td></tr><tr><td>PICSAR197</td><td>4,791,144</td><td>133</td><td>85,113</td><td>4,570</td><td>4,507</td><td>69.33</td><td>ERS6092726</td><td>CAJSOO010000001</td></tr><tr><td>PICSAR198</td><td>4,787,281</td><td>151</td><td>74,155</td><td>4,563</td><td>4,500</td><td>69.32</td><td>ERS6092727</td><td>CAJSON010000001</td></tr><tr><td>PICSAR2</td><td>4,792,264</td><td>156</td><td>77,040</td><td>4,574</td><td>4,511</td><td>69.33</td><td>ERS6092728</td><td>CAJSOW010000001</td></tr><tr><td>PICSAR201</td><td>4,789,399</td><td>170</td><td>68,914</td><td>4,567</td><td>4,504</td><td>69.32</td><td>ERS6092730</td><td>CAJVAW010000001</td></tr><tr><td>PICSAR202</td><td>4,783,908</td><td>160</td><td>64,785</td><td>4,561</td><td>4,498</td><td>69.31</td><td>ERS6092731</td><td>CAJSOX010000001</td></tr><tr><td>PICSAR204</td><td>4,790,713</td><td>139</td><td>86,451</td><td>4,571</td><td>4,508</td><td>69.32</td><td>ERS6092733</td><td>CAJSOV010000001</td></tr><tr><td>PICSAR205</td><td>4,792,146</td><td>154</td><td>74,990</td><td>4,564</td><td>4,501</td><td>69.32</td><td>ERS6377155</td><td>CAJSOP010000001</td></tr></tbody></table>

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<table><caption>TABLE 1 (Continued)</caption><thead><tr><th>Isolate</th><th>Genome size (bp)</th><th>No. of contigs</th><th>N<sub>50</sub> (bp)</th><th>Total no. of genes</th><th>Total no. of CDSs<sup>a</sup></th><th>G+C content (%)</th><th>ENA accession no.</th><th>Contig accession no.</th></tr></thead><tbody><tr><td>PICSAR206</td><td>4,790,443</td><td>140</td><td>73,462</td><td>4,568</td><td>4,505</td><td>69.32</td><td>ERS6092734</td><td>CAJSOT010000001</td></tr><tr><td>PICSAR208</td><td>4,792,845</td><td>147</td><td>73,575</td><td>4,572</td><td>4,509</td><td>69.32</td><td>ERS6092735</td><td>CAJSPD010000001</td></tr><tr><td>PICSAR209</td><td>4,785,508</td><td>185</td><td>62,895</td><td>4,579</td><td>4,516</td><td>69.32</td><td>ERS6092736</td><td>CAJSPG010000001</td></tr><tr><td>PICSAR21</td><td>4,795,268</td><td>165</td><td>71,391</td><td>4,579</td><td>4,516</td><td>69.32</td><td>ERS6377156</td><td>CAJSPA010000001</td></tr><tr><td>PICSAR210</td><td>4,786,061</td><td>170</td><td>64,188</td><td>4,581</td><td>4,518</td><td>69.32</td><td>ERS6092737</td><td>CAJVRD010000001</td></tr><tr><td>PICSAR211</td><td>4,788,728</td><td>133</td><td>83,580</td><td>4,551</td><td>4,488</td><td>69.33</td><td>ERS6092738</td><td>CAJSPB010000001</td></tr><tr><td>PICSAR214</td><td>4,765,345</td><td>213</td><td>56,214</td><td>4,569</td><td>4,506</td><td>69.31</td><td>ERS6092740</td><td>CAJSPC010000001</td></tr><tr><td>PICSAR215</td><td>4,786,273</td><td>194</td><td>53,502</td><td>4,585</td><td>4,522</td><td>69.31</td><td>ERS6092741</td><td>CAJVAT010000001</td></tr><tr><td>PICSAR216</td><td>4,791,256</td><td>124</td><td>97,349</td><td>4,566</td><td>4,503</td><td>69.33</td><td>ERS6092742</td><td>CAJVAU010000001</td></tr><tr><td>PICSAR218</td><td>4,789,693</td><td>123</td><td>92,477</td><td>4,567</td><td>4,504</td><td>69.33</td><td>ERS6092743</td><td>CAJVAV010000001</td></tr><tr><td>PICSAR22</td><td>4,791,855</td><td>151</td><td>79,764</td><td>4,557</td><td>4,494</td><td>69.33</td><td>ERS6092746</td><td>CAJSPF010000001</td></tr><tr><td>PICSAR222</td><td>4,789,088</td><td>183</td><td>60,343</td><td>4,588</td><td>4,525</td><td>69.32</td><td>ERS6092747</td><td>CAJSPE010000001</td></tr><tr><td>PICSAR23</td><td>4,787,284</td><td>142</td><td>92,228</td><td>4,566</td><td>4,503</td><td>69.32</td><td>ERS6092749</td><td>CAJSPH010000001</td></tr><tr><td>PICSAR232</td><td>4,790,364</td><td>144</td><td>73,122</td><td>4,563</td><td>4,500</td><td>69.32</td><td>ERS6092750</td><td>CAJSP010000001</td></tr><tr><td>PICSAR235</td><td>4,781,829</td><td>221</td><td>43,461</td><td>4,598</td><td>4,535</td><td>69.31</td><td>ERS6092751</td><td>CAJSPQ010000001</td></tr><tr><td>PICSAR238</td><td>4,788,942</td><td>173</td><td>64,108</td><td>4,582</td><td>4,520</td><td>69.32</td><td>ERS6092752</td><td>CAJSPP010000001</td></tr><tr><td>PICSAR24</td><td>4,786,946</td><td>159</td><td>77,065</td><td>4,563</td><td>4,500</td><td>69.32</td><td>ERS6092753</td><td>CAJSPK010000001</td></tr><tr><td>PICSAR240</td><td>4,779,977</td><td>168</td><td>62,880</td><td>4,571</td><td>4,508</td><td>69.31</td><td>ERS6092754</td><td>CAJSPN010000001</td></tr><tr><td>PICSAR246</td><td>4,787,832</td><td>195</td><td>58,821</td><td>4,591</td><td>4,528</td><td>69.32</td><td>ERS6092756</td><td>CAJSPR010000001</td></tr><tr><td>PICSAR248</td><td>4,790,004</td><td>160</td><td>69,396</td><td>4,586</td><td>4,523</td><td>69.32</td><td>ERS6092758</td><td>CAJSPJ010000001</td></tr><tr><td>PICSAR249</td><td>4,792,479</td><td>176</td><td>58,262</td><td>4,581</td><td>4,518</td><td>69.32</td><td>ERS6092759</td><td>CAJSPL010000001</td></tr><tr><td>PICSAR25</td><td>4,781,362</td><td>211</td><td>48,503</td><td>4,578</td><td>4,517</td><td>69.31</td><td>ERS6377157</td><td>CAJSPM010000001</td></tr><tr><td>PICSAR250</td><td>4,792,163</td><td>144</td><td>86,432</td><td>4,576</td><td>4,513</td><td>69.32</td><td>ERS6092760</td><td>CAJSPI010000001</td></tr><tr><td>PICSAR252</td><td>4,786,258</td><td>187</td><td>58,262</td><td>4,591</td><td>4,528</td><td>69.32</td><td>ERS6092762</td><td>CAJSPS010000001</td></tr><tr><td>PICSAR253</td><td>4,794,805</td><td>163</td><td>77,033</td><td>4,571</td><td>4,508</td><td>69.31</td><td>ERS6377158</td><td>CAJSPT010000001</td></tr><tr><td>PICSAR254</td><td>4,790,393</td><td>167</td><td>69,690</td><td>4,577</td><td>4,514</td><td>69.33</td><td>ERS6092763</td><td>CAJSPV010000001</td></tr><tr><td>PICSAR255</td><td>4,789,281</td><td>155</td><td>71,960</td><td>4,572</td><td>4,509</td><td>69.33</td><td>ERS6092764</td><td>CAJSPW010000001</td></tr><tr><td>PICSAR26</td><td>4,803,390</td><td>358</td><td>31,565</td><td>4,642</td><td>4,579</td><td>69.3</td><td>ERS6092767</td><td>CAJSPY010000001</td></tr><tr><td>PICSAR28</td><td>4,784,222</td><td>178</td><td>58,791</td><td>4,577</td><td>4,514</td><td>69.31</td><td>ERS6092769</td><td>CAJSPX010000001</td></tr><tr><td>PICSAR29</td><td>4,790,559</td><td>180</td><td>55,537</td><td>4,586</td><td>4,523</td><td>69.32</td><td>ERS6092770</td><td>CAJSPU010000001</td></tr><tr><td>PICSAR3</td><td>4,788,567</td><td>149</td><td>86,432</td><td>4,566</td><td>4,503</td><td>69.33</td><td>ERS6092771</td><td>CAJSPZ010000001</td></tr><tr><td>PICSAR30</td><td>4,788,254</td><td>144</td><td>73,587</td><td>4,558</td><td>4,495</td><td>69.32</td><td>ERS6092772</td><td>CAJSQA010000001</td></tr><tr><td>PICSAR36</td><td>4,788,733</td><td>173</td><td>64,182</td><td>4,578</td><td>4,515</td><td>69.32</td><td>ERS6092773</td><td>CAJSQH010000001</td></tr><tr><td>PICSAR38</td><td>4,783,227</td><td>188</td><td>56,621</td><td>4,589</td><td>4,526</td><td>69.31</td><td>ERS6092774</td><td>CAJSQB010000001</td></tr><tr><td>PICSAR4</td><td>4,789,442</td><td>161</td><td>69,522</td><td>4,572</td><td>4,509</td><td>69.32</td><td>ERS6092775</td><td>CAJSQE010000001</td></tr><tr><td>PICSAR43</td><td>4,795,124</td><td>164</td><td>74,414</td><td>4,570</td><td>4,507</td><td>69.32</td><td>ERS6092776</td><td>CAJSQC010000001</td></tr><tr><td>PICSAR46</td><td>4,789,901</td><td>187</td><td>60,696</td><td>4,595</td><td>4,532</td><td>69.32</td><td>ERS6092777</td><td>CAJSQI010000001</td></tr><tr><td>PICSAR47</td><td>4,788,818</td><td>150</td><td>71,401</td><td>4,572</td><td>4,509</td><td>69.32</td><td>ERS6092778</td><td>CAJSQD010000001</td></tr><tr><td>PICSAR48</td><td>4,785,510</td><td>199</td><td>49,332</td><td>4,578</td><td>4,515</td><td>69.31</td><td>ERS6092779</td><td>CAJSQG010000001</td></tr><tr><td>PICSAR49</td><td>4,781,084</td><td>182</td><td>62,918</td><td>4,585</td><td>4,522</td><td>69.31</td><td>ERS6092780</td><td>CAJSQF010000001</td></tr><tr><td>PICSAR5</td><td>4,784,540</td><td>230</td><td>42,694</td><td>4,594</td><td>4,531</td><td>69.31</td><td>ERS6092781</td><td>CAJSQJ010000001</td></tr><tr><td>PICSAR51</td><td>4,793,239</td><td>189</td><td>59,822</td><td>4,591</td><td>4,528</td><td>69.32</td><td>ERS6092782</td><td>CAJSQL010000001</td></tr><tr><td>PICSAR52</td><td>4,787,441</td><td>152</td><td>72,686</td><td>4,573</td><td>4,510</td><td>69.32</td><td>ERS6092783</td><td>CAJSQM010000001</td></tr><tr><td>PICSAR53</td><td>4,784,438</td><td>179</td><td>58,265</td><td>4,598</td><td>4,535</td><td>69.31</td><td>ERS6092784</td><td>CAJSQN010000001</td></tr><tr><td>PICSAR55</td><td>4,785,207</td><td>218</td><td>40,946</td><td>4,606</td><td>4,543</td><td>69.31</td><td>ERS6092785</td><td>CAJSQK010000001</td></tr><tr><td>PICSAR56</td><td>4,788,148</td><td>166</td><td>68,167</td><td>4,578</td><td>4,515</td><td>69.32</td><td>ERS6092786</td><td>CAJSQP010000001</td></tr><tr><td>PICSAR58</td><td>4,787,822</td><td>178</td><td>64,942</td><td>4,567</td><td>4,504</td><td>69.32</td><td>ERS6092787</td><td>CAJSQQ010000001</td></tr><tr><td>PICSAR6</td><td>4,787,390</td><td>196</td><td>56,620</td><td>4,601</td><td>4,538</td><td>69.32</td><td>ERS6092789</td><td>CAJSQQ010000001</td></tr><tr><td>PICSAR61</td><td>4,787,489</td><td>180</td><td>59,708</td><td>4,572</td><td>4,509</td><td>69.32</td><td>ERS6092791</td><td>CAJSQR010000001</td></tr><tr><td>PICSAR63</td><td>4,790,650</td><td>162</td><td>71,378</td><td>4,572</td><td>4,509</td><td>69.32</td><td>ERS6092792</td><td>CAJSQS010000001</td></tr><tr><td>PICSAR64</td><td>4,787,452</td><td>193</td><td>58,783</td><td>4,580</td><td>4,517</td><td>69.31</td><td>ERS6092793</td><td>CAJSQT010000001</td></tr><tr><td>PICSAR65</td><td>4,782,888</td><td>305</td><td>30,419</td><td>4,639</td><td>4,576</td><td>69.3</td><td>ERS6092794</td><td>CAJSQU010000001</td></tr><tr><td>PICSAR68</td><td>4,786,445</td><td>181</td><td>58,792</td><td>4,565</td><td>4,502</td><td>69.32</td><td>ERS6092796</td><td>CAJSQZ010000001</td></tr><tr><td>PICSAR7</td><td>4,778,202</td><td>248</td><td>36,939</td><td>4,597</td><td>4,534</td><td>69.3</td><td>ERS6092797</td><td>CAJSQV010000001</td></tr><tr><td>PICSAR70</td><td>4,784,997</td><td>142</td><td>92,177</td><td>4,585</td><td>4,522</td><td>69.32</td><td>ERS6092798</td><td>CAJSRA010000001</td></tr><tr><td>PICSAR71</td><td>4,781,870</td><td>222</td><td>48,298</td><td>4,620</td><td>4,557</td><td>69.31</td><td>ERS6092799</td><td>CAJSQX010000001</td></tr><tr><td>PICSAR72</td><td>4,788,047</td><td>175</td><td>59,133</td><td>4,574</td><td>4,511</td><td>69.32</td><td>ERS6092800</td><td>CAJSRC010000001</td></tr><tr><td>PICSAR73</td><td>4,785,412</td><td>174</td><td>64,178</td><td>4,573</td><td>4,510</td><td>69.32</td><td>ERS6092801</td><td>CAJSQY010000001</td></tr><tr><td>PICSAR74</td><td>4,793,447</td><td>159</td><td>66,779</td><td>4,569</td><td>4,506</td><td>69.32</td><td>ERS6092802</td><td>CAJSRB010000001</td></tr><tr><td>PICSAR75</td><td>4,789,467</td><td>174</td><td>66,593</td><td>4,569</td><td>4,506</td><td>69.32</td><td>ERS6092803</td><td>CAJSRD010000001</td></tr><tr><td>PICSAR76</td><td>4,791,598</td><td>148</td><td>82,825</td><td>4,567</td><td>4,504</td><td>69.32</td><td>ERS6092804</td><td>CAJSQW010000001</td></tr><tr><td>PICSAR77</td><td>4,792,094</td><td>147</td><td>86,451</td><td>4,565</td><td>4,502</td><td>69.32</td><td>ERS6092805</td><td>CAJSRF010000001</td></tr></tbody></table>

(Continued on next page)
<table><caption>TABLE 1 (Continued)</caption><thead><tr><th>Isolate</th><th>Genome size (bp)</th><th>No. of contigs</th><th>N<sub>50</sub> (bp)</th><th>Total no. of genes</th><th>Total no. of CDSs<sup>a</sup></th><th>G+C content (%)</th><th>ENA accession no.</th><th>Contig accession no.</th></tr></thead><tbody><tr><td>PICSAR78</td><td>4,787,636</td><td>205</td><td>53,988</td><td>4,592</td><td>4,529</td><td>69.31</td><td>ERS6092806</td><td>CAJSRG010000001</td></tr><tr><td>PICSAR8</td><td>4,782,208</td><td>192</td><td>49,547</td><td>4,565</td><td>4,502</td><td>69.31</td><td>ERS6092807</td><td>CAJSRE010000001</td></tr><tr><td>PICSAR81</td><td>4,787,134</td><td>187</td><td>58,883</td><td>4,563</td><td>4,500</td><td>69.32</td><td>ERS6092808</td><td>CAJSRH010000001</td></tr><tr><td>PICSAR85</td><td>4,785,784</td><td>170</td><td>64,173</td><td>4,577</td><td>4,514</td><td>69.32</td><td>ERS6377159</td><td>CAJSRJ010000001</td></tr><tr><td>PICSAR9</td><td>4,791,470</td><td>127</td><td>85,615</td><td>4,566</td><td>4,503</td><td>69.33</td><td>ERS6092809</td><td>CAJSRI010000001</td></tr><tr><td>PICSAR94</td><td>4,788,723</td><td>179</td><td>64,188</td><td>4,578</td><td>4,515</td><td>69.32</td><td>ERS6092810</td><td>CAJSRK010000001</td></tr><tr><td>PICSAR98</td><td>4,791,377</td><td>137</td><td>85,433</td><td>4,563</td><td>4,500</td><td>69.32</td><td>ERS6092811</td><td>CAJSRL010000001</td></tr><tr><td>PICSAR42</td><td>4,779,801</td><td>229</td><td>50,081</td><td>4,588</td><td>4,526</td><td>69.3</td><td>ERS6377160</td><td>CAJSRM010000001</td></tr><tr><td>PICSAR39</td><td>4,782,076</td><td>212</td><td>47,711</td><td>4,581</td><td>4,518</td><td>69.31</td><td>ERS6377161</td><td>CAJSRN010000001</td></tr><tr><td>PICSAR138</td><td>4,783,258</td><td>239</td><td>40,551</td><td>4,590</td><td>4,527</td><td>69.3</td><td>ERS6377162</td><td>CAJSRO010000001</td></tr><tr><td>PICSAR97</td><td>4,784,339</td><td>215</td><td>46,530</td><td>4,596</td><td>4,533</td><td>69.31</td><td>ERS6377163</td><td>CAJSRP010000001</td></tr><tr><td>PICSAR213</td><td>4,788,192</td><td>152</td><td>80,210</td><td>4,568</td><td>4,505</td><td>69.32</td><td>ERS6377164</td><td>CAJSRR010000001</td></tr><tr><td>PICSAR87</td><td>4,797,579</td><td>167</td><td>66,614</td><td>4,567</td><td>4,504</td><td>69.3</td><td>ERS6377165</td><td>CAJSRQ010000001</td></tr><tr><td>PICSAR171</td><td>4,790,264</td><td>162</td><td>65,791</td><td>4,567</td><td>4,504</td><td>69.31</td><td>ERS6377166</td><td>CAJSRX010000001</td></tr><tr><td>PICSAR200</td><td>4,794,348</td><td>148</td><td>86,432</td><td>4,572</td><td>4,509</td><td>69.32</td><td>ERS6377167</td><td>CAJSRW010000001</td></tr><tr><td>PICSAR236</td><td>4,790,200</td><td>147</td><td>79,765</td><td>4,560</td><td>4,497</td><td>69.33</td><td>ERS6377168</td><td>CAJSRS010000001</td></tr><tr><td>PICSAR35</td><td>4,780,919</td><td>218</td><td>46,619</td><td>4,584</td><td>4,521</td><td>69.31</td><td>ERS6377169</td><td>CAJSRV010000001</td></tr><tr><td>PICSAR190</td><td>4,788,050</td><td>202</td><td>52,943</td><td>4,584</td><td>4,521</td><td>69.3</td><td>ERS6377170</td><td>CAJSRT010000001</td></tr><tr><td>PICSAR144</td><td>4,781,205</td><td>174</td><td>71,974</td><td>4,575</td><td>4,512</td><td>69.32</td><td>ERS6092698</td><td>CAJSRU010000001</td></tr></tbody></table>

were incubated for 16 h at 56°C. DNA was extracted using the NucleoSpin tissue kit (Macherey-Nagel) according to the manufacturer's protocol. DNA was quantified using the QuantiFluor double-stranded DNA (dsDNA) system with the Quantus fluorometer (Promega, France). The extracted DNA (10 to 30 ng) was used to prepare sequencing libraries with the Nextera XT kit (Illumina, San Diego, CA), and sequencing was conducted using the Illumina MiSeq instrument at the @Bridge platform (INRAE, GABI, Jouy en Josas, France). Sequencing was performed in paired-end, 300-bp format and was down-sampled to about 50× coverage. The short-read data were first trimmed using fastp v0.20.1 (5), based on a Phred scale base quality threshold of Q30 and removing reads of less than 21 bp. Kraken2 v2.0.9-beta (6) with the bacteria database was used to perform taxonomic identification at the read level in order to check for potential contamination (Table 1). All reads assigned to the *Mycobacteriaceae* family were extracted and then assembled using SPAdes v3.14 (7) with the "--careful" option. The metrics of the assembled contigs were collected using QUAST v5.0.2 (8), and the contigs were annotated using Prokka v1.14.6 (9) with *M. avium* subsp. *paratuberculosis* K-10 as the reference annotation strain (10, 11). Prodigal (12) was pretrained using the complete genome sequence of strain K-10.

**Data availability.** The sequences were deposited in the European Nucleotide Archive public sequence database under the accession numbers SAMEA8407198 to SAMEA8407344 and SAMEA8692664 to SAMEA8692685 (BioProject accession number PRJEB43898).

## ACKNOWLEDGMENTS

This work was funded by the INRA metaprogram Integrated Management of Animal Health for the GISA-PICSAR project, by APIS-GENE and GDS France for the PARADIGM project, and by APIS-GENE for the Genomap project.

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2. Bannantine JP, Conde C, Bayles DO, Branger M, Biet F. 2020. Genetic diversity among *Mycobacterium avium* subspecies revealed by analysis of complete genome sequences. Front Microbiol 11:1701. https://doi.org/10.3389/fmicb.2020.01701.

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<footnote>a CDSs, coding DNA sequences.</footnote>
<figure><img src="image_3.png" /></figure>

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