9. Remove AMPure XP beads from fridge at least 30 min before
use. Just before use, shake beads vigorously to ensure homogenous and consistent color.
10. Bead pellet is completely dry when fine cracks can be observed.
Do not over dry as this will decrease yield.
11. Remove TapeStation reagents from fridge 30 min prior to use.
12. If sample number is greater than 15, a 96-well plate and
multiple tapes can be used.
13. Using a lower number of cycles will reduce the amount of
duplication in the final sequencing library. The minimum number of cycles required to achieve enough product for hybridization should be determined empirically.
14. There is the potential to run sputum-derived samples with and
without enrichment, for this, the samples would again be
processed in parallel up to the hybridization (Subheading
3.6), but the ‘without enrichment’ samples would forgo the
hybridization step, and move from Subheading 3.5 straight
to 3.7. However, we found that in order to obtain acceptable
depth and coverage of sequencing from sputum sample,
enrichment was absolutely required (see [12] for details).
15. 500–750 ng of DNA from the PreCapture PCR is required for
each hybridization, if there is no sufficient product from one
PCR, repeat the cycling with another 15μL aliquot of the
Adapter Ligated sample, and after cleaning, pool products.
16. If more than 8 samples are being hybridized, use multiple
plates and thermocycler blocks.
17. This step should be performed as quickly as possible to prevent
excessive evaporation.
18. Use new strip caps to seal column 3; the structural integrity of
the caps can be compromised during the previous incubation
steps.
19. Excessive evaporation, such as the sample volume less than
20μL following hybridization, can indicate suboptimal capture
performance.
20. Visually ensure that sample is mixing on the Nutator platform.
21. Prewarm the required volume of SureSelect Wash Buffer #2 at
65
C for at least 1 h before use.
22. Choose index combinations as per Illumina guidelines to
ensure that indexes are balanced.
23. Using a lower number of cycles will reduce the amount of
duplication in the final sequencing library. The number of
cycles required to achieve enough product for sequencing
should be determined empirically.
478
Amanda Claire Brown
use. Just before use, shake beads vigorously to ensure homogenous and consistent color.
10. Bead pellet is completely dry when fine cracks can be observed.
Do not over dry as this will decrease yield.
11. Remove TapeStation reagents from fridge 30 min prior to use.
12. If sample number is greater than 15, a 96-well plate and
multiple tapes can be used.
13. Using a lower number of cycles will reduce the amount of
duplication in the final sequencing library. The minimum number of cycles required to achieve enough product for hybridization should be determined empirically.
14. There is the potential to run sputum-derived samples with and
without enrichment, for this, the samples would again be
processed in parallel up to the hybridization (Subheading
3.6), but the ‘without enrichment’ samples would forgo the
hybridization step, and move from Subheading 3.5 straight
to 3.7. However, we found that in order to obtain acceptable
depth and coverage of sequencing from sputum sample,
enrichment was absolutely required (see [12] for details).
15. 500–750 ng of DNA from the PreCapture PCR is required for
each hybridization, if there is no sufficient product from one
PCR, repeat the cycling with another 15μL aliquot of the
Adapter Ligated sample, and after cleaning, pool products.
16. If more than 8 samples are being hybridized, use multiple
plates and thermocycler blocks.
17. This step should be performed as quickly as possible to prevent
excessive evaporation.
18. Use new strip caps to seal column 3; the structural integrity of
the caps can be compromised during the previous incubation
steps.
19. Excessive evaporation, such as the sample volume less than
20μL following hybridization, can indicate suboptimal capture
performance.
20. Visually ensure that sample is mixing on the Nutator platform.
21. Prewarm the required volume of SureSelect Wash Buffer #2 at
65
C for at least 1 h before use.
22. Choose index combinations as per Illumina guidelines to
ensure that indexes are balanced.
23. Using a lower number of cycles will reduce the amount of
duplication in the final sequencing library. The number of
cycles required to achieve enough product for sequencing
should be determined empirically.
478
Amanda Claire Brown