24. We have found it to be more accurate to use the TapeStation
data to establish the size of the DNA fragments and the Qubit
to measure the DNA concentration in order to calculate the
molarity.
25. Calculate molarity of each Post-Index Amplified sample as.
μg DNA Â
pmol
660 pg
Â
10
6 pg
1 μg
Â
1
N
¼ pM DNA,
where N is the average fragment size and
pmol
660 pg is the
average molecular weight of a nucleotide pair.
26. For creating a sample sheet in Excel, see https://support.
illumina.com/content/dam/illumina-support/documents/
documentation/system_documentation/miseq/miseq-sam
ple-sheet-quick-ref-guide-15028392-j.pdf for the parameters
required.
27. PhiX control is helpful when sequencing low diversity samples;
however, the choice to add it as a spike in is down to the
individual user. We found that it improved our cluster
formation.
28. If you are using v3 chemistry, then further dilution to 12.5 pM
is not required.
29. Denatured PhiX libraries can be stored for up to 3 weeks at
À20
C.
30. A 300-cycle run takes ~24 h to complete and monitor run for
cluster number and performance.
Acknowledgements
The PATHSEEK consortium received funding from the European
Union’s Seventh Programme for research, technological development, and demonstration under grant agreement No 304875. We
acknowledge all the help from the other members of the consortium involved with the establishment of this methodology, particular thanks go to Mette Christensen and Helena Tutill, UCL.
References
1. Ko ¨ser CU, Ellington MJ, Cartwright EJP et al
(2012) Routine use of microbial whole
genome sequencing in diagnostic and public
health microbiology. PLoS Pathog 8:
e1002824
2. Ko ¨ser CU, Bryant JM, Becq J et al (2013)
Whole-genome sequencing for rapid susceptibility testing of M. tuberculosis. N Engl J Med
369:290–292
3. Olsen RJ, Long SW, Musser JM (2012) Bacterial genomics in infectious disease and the clinical pathology laboratory. Arch Pathol Lab
Med 136:1414–1422
4. https://www.who.int/mediacentre/news/
releases/2010/tb_test_20101208/en/
5. Helb D, Jones M, Story E, Boehme C,
Wallace E, Ho K, Kop J, Owens MR,
Rodgers R, Banada P, Safi H, Blakemore R,
Direct WGS of M. tb’
479
data to establish the size of the DNA fragments and the Qubit
to measure the DNA concentration in order to calculate the
molarity.
25. Calculate molarity of each Post-Index Amplified sample as.
μg DNA Â
pmol
660 pg
Â
10
6 pg
1 μg
Â
1
N
¼ pM DNA,
where N is the average fragment size and
pmol
660 pg is the
average molecular weight of a nucleotide pair.
26. For creating a sample sheet in Excel, see https://support.
illumina.com/content/dam/illumina-support/documents/
documentation/system_documentation/miseq/miseq-sam
ple-sheet-quick-ref-guide-15028392-j.pdf for the parameters
required.
27. PhiX control is helpful when sequencing low diversity samples;
however, the choice to add it as a spike in is down to the
individual user. We found that it improved our cluster
formation.
28. If you are using v3 chemistry, then further dilution to 12.5 pM
is not required.
29. Denatured PhiX libraries can be stored for up to 3 weeks at
À20
C.
30. A 300-cycle run takes ~24 h to complete and monitor run for
cluster number and performance.
Acknowledgements
The PATHSEEK consortium received funding from the European
Union’s Seventh Programme for research, technological development, and demonstration under grant agreement No 304875. We
acknowledge all the help from the other members of the consortium involved with the establishment of this methodology, particular thanks go to Mette Christensen and Helena Tutill, UCL.
References
1. Ko ¨ser CU, Ellington MJ, Cartwright EJP et al
(2012) Routine use of microbial whole
genome sequencing in diagnostic and public
health microbiology. PLoS Pathog 8:
e1002824
2. Ko ¨ser CU, Bryant JM, Becq J et al (2013)
Whole-genome sequencing for rapid susceptibility testing of M. tuberculosis. N Engl J Med
369:290–292
3. Olsen RJ, Long SW, Musser JM (2012) Bacterial genomics in infectious disease and the clinical pathology laboratory. Arch Pathol Lab
Med 136:1414–1422
4. https://www.who.int/mediacentre/news/
releases/2010/tb_test_20101208/en/
5. Helb D, Jones M, Story E, Boehme C,
Wallace E, Ho K, Kop J, Owens MR,
Rodgers R, Banada P, Safi H, Blakemore R,
Direct WGS of M. tb’
479