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    NGS CF Control Panel G211:
 
Product Name

BACKGROUND:
Cystic fibrosis (CF) is the most common inherited disease in the United States today. CF is an autosomal recessive disorder, caused by the presence of mutations in both copies of the gene for the protein, cystic fibrosis transmembrane conductance regulator (CFTR). CF has a disease occurrence of approximately one out of 2,000 – 4,000 live births1. Although CF does not affect all people the same way, it is generally recognized as causing persistent lung infections. CF also affects other organs, including the pancreas, liver, kidneys and intestine. It is characterized by the buildup of thick, sticky mucus in the lungs, pancreas and other organs, resulting in a complex multi-organ, multisystem disease1.  There is no cure for CF but some therapies may prolong life, therefore, a key focus of current interventional efforts is early diagnosis through newborn screening2.

CFTR protein is involved in production of sweat, digestive fluids and mucus. The gene was first identified in 1989 on human chromosome 7 and contains 27 coding exons3. When CFTR is not functional, these secretions which are usually thin and watery instead become thick and mucus-like and ultimately lead to nutritional decline and progression of lung disease associated with morbidity and mortality1. Over 2,000 variants have been reported in the CFTR coding and flanking sequence, in all ethnic and racial populations at various frequencies4. However, less than 10% of these reported variants have been interpreted to be clinically-relevant variants. The 187 CFTR variants listed in Table 1 are contained in the 6 tubes of NGS CF Control Panel G211. The 143 asterisked (*) variants have been identified as CF-causing by The Clinical and Functional Translation of CFTR (CFTR2) international project and are listed in the CFTR2 database at http://cftr2.org (August 13, 2015 version)5,6. Included in Table 1 are the 23 mutations recommended in 2004 by the American College of Medical Genetic (ACMG) and in 2011 by the American College of Obstetricians and Gynecologists (ACOG)7,8. These mutations have been bolded and asterisked in Table 1. Legacy nomenclature is used throughout this Package Insert.

INTENDED USE:
The NGS CF Control Panel G211 is intended for in vitro use as a quality control to monitor the analytical performance of library preparation, sequencing and data analysis associated with Next Generation Sequencing for identifying variants within the CFTR gene. Additionally, because the NGS CF Control Panel G211 carries multiple insertions, deletions and homopolymers of varying lengths and composition, this control panel can also serve as a method control to monitor the ability of any NGS test system to correctly identify these types of variants. Appropriate primers are required to amplify the areas of interest.

NGS CF Control Panel G211 is provided for Research Use Only (RUO). It cannot be cloned, sold, or transferred without the explicit written consent of MMQCI.

PRODUCT SUMMARY AND PRINCIPLE:
Next Generation Sequencing (NGS) technology, also known as massively parallel sequencing, involves sequencing of spatially separated, clonally amplified DNA templates to generate simultaneous sequencing of thousands to millions of relatively short nucleic acid sequences in parallel. This technology allows for targeted, resequencing of the CFTR gene and includes a complex multistep procedure for library preparation, target enrichment and sequencing analysis.

NGS CF Control Panel G211 is designed as a multiplex reference material to monitor the simultaneous detection of the CFTR mutations and variants associated with cystic fibrosis listed in Table 1, including the 23 mutations recommended by ACMG and ACOG. The NGS CF Control Panel G211 product is intended to be analyzed routinely with each cystic fibrosis test run and provide a quality control for all aspects of library preparation, sequencing, and data analysis.

Controls should be tested routinely as a matter of Good Laboratory Practice and according to guidelines or requirements of local, state, and/or federal regulations or accrediting organizations. Best practice is to establish a quality control program for every assay performed by the laboratory9, 10. Routine use of quality controls that are consistent lot to lot assists the laboratory in identifying shifts, trends, and increased frequency of random errors caused by variations in the test system. Early investigation can prevent failed assay runs.

COMPOSITION:
NGS CF Control Panel G211 consists of synthetic CFTR DNA suspended in a non-infectious buffer with stabilizers. The synthetic DNA is composed of all 27 CFTR gene exons plus intronic borders, and contains CF associated mutations and variants divided among 6 tubes (G211A, G211B, G211C, G211D, G211E and G211F). The mutations and variants present in NGS CF Control Panel G211 are described in Table 1. This control panel is provided as a non-extractable control with copy numbers of target sequence that are similar to that of extracted human whole blood samples.

INSTRUCTIONS FOR USE:
Controls are ready to use; no extraction is needed.

  1. Thaw all controls completely.
  2. Allow the controls to be tested to come to room temperature (18° – 25°C).
  3. Mix well by flicking the control tubes and vortex immediately before using.
  4. Quick spin to remove droplets from the tube cap before opening.
  5. Pipette the same volume (usually 5-10uL) pipetted for patient sample DNA for each control tube, and treat just as you would extracted genomic DNA.
  6. Follow all other procedures as described by test manufacturer’s product insert.
  7. Opened material should be tightly capped and returned to freezer (< -20oC) immediately after use.

Note: NGS CF Control Panel G211 cannot be quantified or measured by standard methods such as spectrophotometry (A260nm/A280nm) because synthetic DNA has significantly less mass than genomic DNA. However, the controls are manufactured to have approximately equivalent copy numbers of the target gene as genomic DNA. Please contact MMQCI if guidance on volume to add to assay is desired.

STORAGE and STABILITY:
Upon receipt and after opening, the material should be stored at < -20°C. Unopened NGS CF Control Panel G211 is stable through the expiration date printed on each tube when stored frozen (< -20°C). Opened material should be tightly capped and returned to the freezer (< -20°C) immediately after use. The material is stable for five freeze/ thaw cycles.

PRECAUTIONS and WARNINGS:

  • Do not dilute.
  • Do not transfer control material to a new tube.
  • Do not quantify by spectrophotometry or other standard methods.
  • Do not freeze/ thaw more than 5 times.
  • This product does not contain any biological material of human origin and is not infectious. Universal Precautions are not required when handling this product.

EXPECTED VALUES:
The genotype for each CFTR variant found in tubes A-F of NGS CF Control Panel G211 is listed in Table 1. The sequence of NGS CF Control Panel G211 has been confirmed by bi-directional Sanger sequencing. In order for an NGS assay to detect all genotypes listed, the NGS assay primers must have sufficient control sequence on either side of the variant to anneal and amplify the area of interest. If unexpected calls are reported, or variants not detected, your laboratory’s NGS primers may require sequence not present in NGS CF Control Panel G211, or they may anneal poorly due to the presence of a mutation. For example, S489X HET is present in Tube G211F but not detected by the MiSeqDx™ CF 139-Variant Assay because the forward primer aligns across 2 mutations, 1525-1G>A and M470V, and does not anneal well enough to amplify its amplicon sufficiently. Please contact MMQCI for interpretation assistance as needed.

The laboratory should follow Good Laboratory Practice (GLP) and establish its own performance characteristics for NGS CF Control Panel G211 in demonstrating adequate system performance.

ORDERING INFORMATION:
NGS CF Control Panel G211
Part Number: G211
Kit Contains:   6 tubes x 50 µL
Order and Customer Support: Email: info@mmqci.com, Phone: 207-885-1072 ext1

Maine Molecular Quality Controls, Inc.
23 Mill Brook Road
Saco, ME  04072 USA
Phone: 207-885-1072, FAX: 207-885-1079, Web:  www.mmqci.com

References:

  1. Moskowitz SM, Chmiel JF, Sternan DL, Cheng E, Gibson RL et al. (2008) Clinical practice and genetic counseling for cystic fibrosis and CFTR-related disorders. Genetics of Medicine 10(12):851-868.
  2. The Leeds Method of Management. April, 2008. Screening newborn infants for Cystic Fibrosis [online]. Leeds Regional Adult and Paediatric Cystic Fibrosis Units, St James's University Hospital, Leeds, UK. Available from http://www.cysticfibrosismedicine.com.
  3. Pseudomonas Genome Project, "Cystic Fibrosis." Obtained from the WWW 10/31/98: http://www.pseudomonas.com/cystic_fibrosis.html
  4. Cystic Fibrosis Mutation Database (CFTR1). Available at www.genet.sickkids.on.ca/app. (August 2013).
  5. Clinical and Functional Translation of CFTR (CFTR2). Available at www.cftr2.org. (August 2015).
  6. Sosnay PR, Siklosi Kr, Van Goor F, Kaniecki K, Yu H, et al. (2013) Defining the disease liability of variants in the cystic fibrosis transmembrane conductance regulator gene. Nat Genet. 45(10).
  7. Watson MS, Cutting GR, Desnick RJ, Driscoll DA, Klinger K et al. (2004) Cystic fibrosis population carrier screening : 2004 revision of American College of Medical Genetics mutation panel. Genetics in Medicine 6(5):387-391.
  8. Committee on Genetics. (April 2011) The American College of Obstetricians and Gynecologists Committee Mutations-Correlation with Incidence Data and Application to Screening. Human Mutation 19:575-606.
  9. ISO 15189: Medical laboratories – Particular requirements for quality and competence.
  10. CAP Molecular Pathology Checklist; Commission on Laboratory Accreditation, Laboratory Accreditation Program, Mol.20000.

Table 1. NGS CF Control Panel G211. Variants are listed in genomic coordinate order. Bold = ACMG recommended panel of 23 variants.

Asterisk* = clinically relevant CFTR2 variants.

CFTR Mutation

Legacy Name

Tube

G211A

Tube

G211B

Tube

G211C

Tube

G211D

Tube

G211E

Tube

G211F

1. M1V*

WT

WT

WT

WT

WT

het

2. R31C

WT

WT

het

WT

WT

WT

3. CFTR dele2,3*

het

WT

WT

WT

WT

WT

4. Q39X*

WT

WT

WT

WT

WT

het

5. 296+2T>A

WT

WT

WT

WT

het

WT

6. E60X*

WT

het

WT

WT

WT

WT

7. P67L*

WT

WT

WT

WT

het

WT

8. R74W

WT

WT

WT

WT

WT

het

9. R75X*

WT

WT

WT

het

WT

WT

10. G85E*

WT

het

WT

WT

WT

WT

11. 394delTT*

WT

WT

het

WT

WT

WT

12. 405+1 G>A*

WT

WT

WT

WT

het

WT

13. 405+3A>C*

WT

WT

WT

het

WT

WT

14. 406-1 G>A*

WT

WT

WT

het

WT

WT

15. E92K*

WT

WT

WT

WT

WT

HET

16. E92XG>T*

WT

WT

WT

WT

het

WT

17. Q98X*

WT

WT

het

WT

WT

WT

18. 444delA*

WT

WT

WT

het

WT

WT

19. 457TAT->G*

WT

WT

WT

WT

het

WT

20. D110H*

WT

WT

WT

WT

WT

het

21. R117C*

WT

WT

WT

het

WT

WT

22. R117H

WT

WT

het

WT

WT

WT

23. Y122X*

het

WT

WT

WT

WT

WT

24. 574delA*

WT

WT

WT

WT

het

WT

25. I148T

WT

het

WT

WT

WT

WT

26. 621+1 G>T*

WT

het

WT

WT

WT

het

27. 663delT*

WT

WT

WT

WT

het

WT

28. G178R*

WT

WT

WT

het

WT

WT

29. 711+1 G>T*

WT

het

WT

WT

WT

WT

30. 711+3 A>G*

WT

WT

WT

WT

WT

het

31. 711+5 G>A*

WT

WT

het

WT

WT

WT

32. 712-1 G>T*

WT

WT

WT

WT

het

WT

33. H199Y

WT

HET

WT

WT

WT

WT

34. P205S C>T*

WT

WT

het

WT

WT

WT

35. L206W*

WT

WT

WT

het

WT

WT

36. Q220X*

WT

WT

WT

WT

WT

het

37. L227R*

WT

WT

het

WT

WT

WT

38. 852del22*

WT

WT

het

WT

WT

WT

39. 935delA*

WT

WT

WT

het

WT

WT

40. 936delTA

WT

WT

WT

WT

het

WT

41. delF311

WT

WT

WT

het

WT

WT

42. 1078delT*

WT

het

WT

WT

WT

WT

43. G330X*

WT

WT

WT

het

WT

WT

44. R334W*

WT

het

WT

WT

WT

WT

45. I336K*

WT

WT

WT

WT

WT

het

46. T338IC>T*

WT

WT

WT

WT

het

WT

47. S341P*

WT

WT

het

WT

WT

WT

48. 1154insTC*

het

WT

WT

WT

WT

WT

49. R347H*

WT

WT

het

WT

WT

WT

50. R347P*

WT

het

WT

WT

WT

WT

51. R352Q*

WT

WT

WT

het

WT

WT

52. 1213delT*

WT

WT

WT

WT

WT

het

53. S364P

WT

WT

WT

het

WT

WT

54. 1248+1G>A*

het

WT

WT

WT

WT

WT

55. 1259insA*

WT

WT

WT

WT

WT

het

56. 1288insTA*

WT

WT

WT

WT

het

WT

57. W401X (TAG)*

het

WT

WT

WT

WT

WT

58. W401X (TGA)*

WT

WT

WT

het

WT

WT

59. 1341+1G>A*

WT

WT

het

WT

WT

WT

60. PolyT

7T/7T

9T/9T

7T/5T

5T/9T

9T

9T/9T

61. 1461ins4*

WT

WT

WT

WT

WT

het

62. A455E*

WT

het

WT

WT

WT

WT

63. 1525-1G>A*

WT

WT

WT

WT

WT

het

64. S466X (C>A)

WT

WT

WT

WT

HET

WT

65. S466X (C>G)*

het

WT

WT

WT

WT

WT

66. L467P*

WT

WT

het

WT

WT

WT

67. M470V

hET

HOM

hom

HOM

hom

hom

68. 1548delG*

WT

WT

WT

het

WT

WT

69. G480C

WT

WT

WT

het

WT

WT

70. S489X*

WT

WT

WT

WT

WT

het

71. S492F*

het

WT

WT

WT

WT

WT

72. Q493X*

het

WT

WT

WT

WT

WT

73. I506V

WT

WT

HET

WT

WT

WT

74. I506T (T>C)

WT

WT

WT

WT

het

WT

75. I507V

HET

WT

WT

WT

WT

WT

76. I507del*

WT

WT

het

het

WT

WT

77. F508del*

WT

het

WT

WT

WT

WT

78. F508C

HET

WT

WT

WT

WT

WT

79. 1677delTA*

WT

WT

het

het

WT

WT

80. V520F*

WT

het

WT

WT

WT

WT

81. Q525X*

WT

WT

WT

WT

WT

het

82. 1717-1G>A*

WT

het

WT

WT

WT

WT

83. 1717-8G>A*

WT

WT

WT

WT

WT

het

84. G542X*

WT

het

WT

WT

WT

WT

85. S549R (c.1645A>C)*

WT

WT

het

WT

WT

WT

86. S549R (c.1647T>G)*

het

WT

WT

WT

WT

WT

87. S549N*

WT

WT

WT

het

WT

WT

88. G551D*

WT

het

WT

WT

WT

WT

89. Q552XC>T*

WT

WT

WT

WT

het

WT

90. R553X*

WT

WT

het

WT

WT

WT

91. L558S

WT

WT

WT

WT

WT

het

92. A559T*

het

WT

WT

WT

WT

WT

93. R560T*

WT

het

WT

WT

WT

WT

94. R560K*

WT

WT

WT

het

WT

WT

95. 1812-1 G>A*

WT

WT

WT

het

WT

WT

96. Y569D*

WT

WT

WT

WT

WT

het

97. G576A

WT

WT

WT

het

WT

WT

98. D579G

WT

WT

WT

WT

het

WT

99. E585X*

WT

WT

het

WT

WT

WT

100. 1898+1 G>A*

WT

het

WT

WT

WT

WT

101. 1898+3 A>G*

WT

WT

WT

WT

het

WT

102. 1898+5G>T

het

WT

WT

WT

WT

WT

103. 1949del84

het

WT

WT

WT

WT

WT

104. D614G*

WT

WT

WT

WT

WT

het

105. G622D

WT

WT

WT

het

WT

WT

106. 2055del9>A*

WT

WT

WT

het

WT

WT

107. 2108delA

WT

WT

WT

WT

het

WT

108. R668C

WT

WT

WT

WT

WT

het

109. 2143delT*

WT

het

WT

WT

WT

WT

110. 2183AA>G*

WT

WT

het

WT

WT

WT

111. 2184insA*

WT

WT

WT

WT

HET

WT

112. 2184delA*

WT

het

WT

WT

WT

WT

113. R709XC>T*

WT

WT

WT

WT

het

WT

114. K710X*

WT

WT

WT

het

WT

WT

115. 2307insA*

het

WT

WT

WT

WT

WT

116. L732X*

WT

WT

WT

WT

WT

het

117. 2347delG*

WT

WT

WT

het

WT

WT

118. V754M

WT

WT

WT

WT

WT

het

119. R764XC>T*

WT

WT

WT

WT

het

WT

120. 2585delT*

WT

WT

WT

WT

WT

het

121. E822X*

WT

WT

WT

het

WT

WT

122. 2622+1G>A*

WT

het

WT

WT

WT

WT

123. E831X*

WT

WT

WT

WT

WT

het

124. W846X*

het

WT

WT

WT

WT

WT

125. W846X(2670)

WT

het

WT

WT

WT

WT

126. R851X*

WT

WT

WT

WT

WT

het

127. 2711delT*

WT

WT

WT

het

WT

WT

128. 2789+2insA

WT

WT

WT

WT

WT

het

129. 2789+5G>A*

WT

het

WT

WT

WT

WT

130. Q890X*

WT

WT

WT

het

WT

het

131. 2869insG*

WT

WT

WT

het

WT

WT

132. L927P*

WT

WT

WT

WT

WT

het

133. S945L*

WT

WT

WT

WT

WT

het

134. 3007delG*

WT

het

WT

WT

WT

WT

135. G970R*

WT

WT

WT

WT

WT

het

136. S977F

WT

WT

WT

WT

WT

het

137. 3120G>A*

WT

WT

WT

het

WT

WT

138. 3120+1G>A*

WT

het

WT

WT

WT

WT

139. 3121-1G>A*

WT

WT

WT

WT

WT

het

140. L997F (G>C)

WT

WT

WT

WT

het

WT

141. 3171delC*

WT

WT

WT

WT

het

WT

142. 3199del6

WT

het

WT

WT

WT

WT

143. I1027T

WT

WT

WT

WT

WT

het

144. 3272-26A>G*

WT

WT

het

WT

WT

WT

145. F1052V

WT

WT

WT

WT

WT

het

146. L1065P*

WT

WT

WT

WT

het

WT

147. R1066C*

WT

WT

WT

het

WT

WT

148. R1066H*

WT

WT

het

WT

WT

WT

149. G1069R

WT

WT

WT

WT

WT

het

150. R1070W

WT

het

WT

WT

WT

WT

151. R1070Q

het

WT

WT

WT

WT

WT

152. L1077P*

WT

het

WT

WT

het

WT

153. W1089X*

WT

WT

WT

het

WT

WT

154. Y1092X (C>A)*

het

WT

WT

WT

WT

WT

155. Y1092X (C>G)*

WT

WT

het

WT

WT

WT

156. M1101K*

Het

WT

WT

WT

WT

WT

157. E1104X*

WT

WT

WT

WT

WT

het

158. D1152H

WT

het

WT

WT

WT

WT

159. R1158X*

WT

WT

WT

het

WT

WT

160. R1162X*

WT

het

WT

WT

WT

WT

161. R1162L

WT

WT

WT

WT

WT

het

162. 3659delC*

WT

het

WT

WT

WT

WT

163. 3667del4

WT

WT

WT

WT

het

WT

164. S1196X*

WT

WT

WT

het

WT

WT

165. W1204X (3743G>A)*

WT

WT

WT

WT

het

WT

166. W1204X (3744G>A)*

WT

WT

WT

WT

WT

het

167. 3791delC

WT

WT

WT

het

WT

WT

168. I1234V*

WT

WT

WT

WT

WT

het

169. S1235R

WT

het

WT

WT

WT

WT

170. Q1238XC>T

WT

WT

WT

WT

het

WT

171. 3849+10kb C>T*

WT

het

WT

WT

WT

WT

172. 3849+4A>G*

het

WT

WT

WT

WT

WT

173. G1244E*

WT

WT

WT

WT

het

het

174. 3876delA*

WT

het

WT

WT

WT

WT

175. S1251N*

het

WT

WT

WT

hET

WT

176. S1255XA>G

WT

WT

het

WT

WT

WT

177. S1255XC>A*

WT

WT

het

WT

WT

WT

178. 3905insT*

WT

het

WT

WT

WT

WT

179. D1270N

WT

WT

WT

het

WT

WT

180. W1282X*

WT

het

WT

WT

WT

WT

181. 4005+1G>A*

WT

WT

WT

WT

WT

het

182. 4016insT*

WT

WT

WT

WT

het

WT

183. N1303K*

WT

het

WT

WT

WT

het

184. Q1313X*

het

WT

WT

WT

WT

WT

185. CFTRdel22,23*

WT

WT

WT

WT

WT

het

186. 4209TGTT>AA*

het

WT

WT

WT

WT

WT

187. 4382delA*

HET

WT

WT

WT

WT

WT

 

Safety Data Sheet

 


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