13. Sheared DNA is characterized by a smear located between
200 and 600 bp.
14. Calculate the volume corresponding to 500 ng of DNA with
the following equation: volume in μl ¼ (500 ng)/(DNA concentration in ng/μl).
15. The library should show a smear in which most of the PCR
products are between 200 and 600 bp and no amplification for
the control reaction.
16. Calculate the volume corresponding to 15 or 20 ng with the
following equation: volume in μl ¼ (DNA concentration of
library in ng/ml)/(15 or 20 ng).
17. DNA concentration and volume required for sequencing vary
depending on the sequencing platform.
References
1. Picardeau M (2018) Toolbox of molecular
techniques for studying Leptospira spp. Curr
Top Microbiol Immunol 415:141–162.
https://doi.org/10.1007/82_2017_45
2. Murray GL, Morel V, Cerqueira GM, Croda J,
Srikram A, Henry R, Ko AI, Dellagostin OA,
Bulach DM, Sermswan RW, Adler B, Picardeau
M (2009) Genome-wide transposon mutagenesis in pathogenic Leptospira species. Infect
Immun 77(2):810–816. https://doi.org/10.
1128/IAI.01293-08
3. van Opijnen T, Bodi KL, Camilli A (2009)
Tn-seq: high-throughput parallel sequencing
for fitness and genetic interaction studies in
microorganisms.
Nat
Methods
6
(10):767–772.
https://doi.org/10.1038/
nmeth.1377
4. van Opijnen T, Dw L, Camilli A (2015)
Genome-wide fitness and genetic interactions
determined by Tn-seq, a high-throughput
massively parallel sequencing method for
microorganisms. Curr Protoc Microbiol
36:1E.3.1–1E.324.
https://doi.org/10.
1002/9780471729259.mc01e03s36
5. Troy EB, Lin T, Gao L, Lazinski DW,
Camilli A, Norris SJ, Hu LT (2013) Understanding barriers to Borrelia burgdorferi dissemination during infection using massively
parallel sequencing. Infect Immun 81
(7):2347–2357. https://doi.org/10.1128/
IAI.00266-13
6. Lourdault K, Matsunaga J, Haake DA (2016)
High-throughput parallel sequencing to measure fitness of Leptospira interrogans transposon insertion mutants during acute infection.
PLoS Negl Trop Dis 10(11):e0005117.
https://doi.org/10.1371/journal.pntd.
0005117
7. Lourdault K, Matsunaga J, Evangelista KV,
Haake DA (2017) High-throughput parallel
sequencing to measure fitness of Leptospira
interrogans transposon insertion mutants during golden Syrian hamster infection. J Vis Exp
(130):56442.
https://doi.org/10.3791/
56442
8. Ellinghausen HC Jr, McCullough WG (1965)
Nutrition of Leptospira pomona and growth of
13 other serotypes: fractionation of oleic albumin complex and a medium of bovine albumin
and polysorbate 80. Am J Vet Res 26:45–51
9. Johnson RC, Harris VG (1967) Differentiation
of
pathogenic
and
saprophytic
letospires. I. Growth at low temperatures. J
Bacteriol 94(1):27–31
10. Slamti L, Picardeau M (2012) Construction of
a library of random mutants in the spirochete
Leptospira biflexa using a mariner transposon.
Methods Mol Biol 859:169–176. https://doi.
org/10.1007/978-1-61779-603-6_9
11. Wunder EA Jr, Figueira CP, Santos GR,
Lourdault K, Matthias MA, Vinetz JM,
Ramos E, Haake DA, Picardeau M, Dos Reis
MG, Ko AI (2016) Real-time PCR reveals
rapid dissemination of Leptospira interrogans
after intraperitoneal and conjunctival inoculation of hamsters. Infect Immun 84
(7):2105–2115. https://doi.org/10.1128/
iai.00094-16
12. Cameron CE (2015) Leptospiral structure,
physiology, and metabolism. Curr Top Microbiol Immunol 387:21–41. https://doi.org/
10.1007/978-3-662-45059-8_3
106
Kristel Lourdault and James Matsunaga
200 and 600 bp.
14. Calculate the volume corresponding to 500 ng of DNA with
the following equation: volume in μl ¼ (500 ng)/(DNA concentration in ng/μl).
15. The library should show a smear in which most of the PCR
products are between 200 and 600 bp and no amplification for
the control reaction.
16. Calculate the volume corresponding to 15 or 20 ng with the
following equation: volume in μl ¼ (DNA concentration of
library in ng/ml)/(15 or 20 ng).
17. DNA concentration and volume required for sequencing vary
depending on the sequencing platform.
References
1. Picardeau M (2018) Toolbox of molecular
techniques for studying Leptospira spp. Curr
Top Microbiol Immunol 415:141–162.
https://doi.org/10.1007/82_2017_45
2. Murray GL, Morel V, Cerqueira GM, Croda J,
Srikram A, Henry R, Ko AI, Dellagostin OA,
Bulach DM, Sermswan RW, Adler B, Picardeau
M (2009) Genome-wide transposon mutagenesis in pathogenic Leptospira species. Infect
Immun 77(2):810–816. https://doi.org/10.
1128/IAI.01293-08
3. van Opijnen T, Bodi KL, Camilli A (2009)
Tn-seq: high-throughput parallel sequencing
for fitness and genetic interaction studies in
microorganisms.
Nat
Methods
6
(10):767–772.
https://doi.org/10.1038/
nmeth.1377
4. van Opijnen T, Dw L, Camilli A (2015)
Genome-wide fitness and genetic interactions
determined by Tn-seq, a high-throughput
massively parallel sequencing method for
microorganisms. Curr Protoc Microbiol
36:1E.3.1–1E.324.
https://doi.org/10.
1002/9780471729259.mc01e03s36
5. Troy EB, Lin T, Gao L, Lazinski DW,
Camilli A, Norris SJ, Hu LT (2013) Understanding barriers to Borrelia burgdorferi dissemination during infection using massively
parallel sequencing. Infect Immun 81
(7):2347–2357. https://doi.org/10.1128/
IAI.00266-13
6. Lourdault K, Matsunaga J, Haake DA (2016)
High-throughput parallel sequencing to measure fitness of Leptospira interrogans transposon insertion mutants during acute infection.
PLoS Negl Trop Dis 10(11):e0005117.
https://doi.org/10.1371/journal.pntd.
0005117
7. Lourdault K, Matsunaga J, Evangelista KV,
Haake DA (2017) High-throughput parallel
sequencing to measure fitness of Leptospira
interrogans transposon insertion mutants during golden Syrian hamster infection. J Vis Exp
(130):56442.
https://doi.org/10.3791/
56442
8. Ellinghausen HC Jr, McCullough WG (1965)
Nutrition of Leptospira pomona and growth of
13 other serotypes: fractionation of oleic albumin complex and a medium of bovine albumin
and polysorbate 80. Am J Vet Res 26:45–51
9. Johnson RC, Harris VG (1967) Differentiation
of
pathogenic
and
saprophytic
letospires. I. Growth at low temperatures. J
Bacteriol 94(1):27–31
10. Slamti L, Picardeau M (2012) Construction of
a library of random mutants in the spirochete
Leptospira biflexa using a mariner transposon.
Methods Mol Biol 859:169–176. https://doi.
org/10.1007/978-1-61779-603-6_9
11. Wunder EA Jr, Figueira CP, Santos GR,
Lourdault K, Matthias MA, Vinetz JM,
Ramos E, Haake DA, Picardeau M, Dos Reis
MG, Ko AI (2016) Real-time PCR reveals
rapid dissemination of Leptospira interrogans
after intraperitoneal and conjunctival inoculation of hamsters. Infect Immun 84
(7):2105–2115. https://doi.org/10.1128/
iai.00094-16
12. Cameron CE (2015) Leptospiral structure,
physiology, and metabolism. Curr Top Microbiol Immunol 387:21–41. https://doi.org/
10.1007/978-3-662-45059-8_3
106
Kristel Lourdault and James Matsunaga