Hello all,
I've searched the threads but have not found an answer so hopefully I'm not repeating an earlier question.
In the past, DNA extraction from several slides was necessary to get sufficient material for sequencing. However, the amount of material required is less and less, and we are now getting enough material from 1-2 slides for the 60X coverage we want. This brings up the question of whether we "should" do the library prep separately from different slides and/or sequence them separately (for instance, this would generate 2 output files at 30X each) - this would give us the option to compare the 2 output files for differences (that could be technical error or biological differences between 2 slides from the same tumor).
Our team has debated the best approach and are trying to choose between several methods:
1. If there is sufficient material, sequence the DNA from different slides separately and combine the data from each 30X to get 60X coverage.
2. Combine the DNA from both slides and take the right sized sample for library prep (and save the rest).
3. Do library prep separately then combine libraries for sequencing.
I think that we have a good handle on the technical pros and cons to each approach, my question is whether there is a standard practice in the industry? The lab we work with hasn't changed their protocol (that is they still combine DNA prior to library prep), but we have the opportunity to have them do it differently for our next experiments.
Thanks!!
I've searched the threads but have not found an answer so hopefully I'm not repeating an earlier question.
In the past, DNA extraction from several slides was necessary to get sufficient material for sequencing. However, the amount of material required is less and less, and we are now getting enough material from 1-2 slides for the 60X coverage we want. This brings up the question of whether we "should" do the library prep separately from different slides and/or sequence them separately (for instance, this would generate 2 output files at 30X each) - this would give us the option to compare the 2 output files for differences (that could be technical error or biological differences between 2 slides from the same tumor).
Our team has debated the best approach and are trying to choose between several methods:
1. If there is sufficient material, sequence the DNA from different slides separately and combine the data from each 30X to get 60X coverage.
2. Combine the DNA from both slides and take the right sized sample for library prep (and save the rest).
3. Do library prep separately then combine libraries for sequencing.
I think that we have a good handle on the technical pros and cons to each approach, my question is whether there is a standard practice in the industry? The lab we work with hasn't changed their protocol (that is they still combine DNA prior to library prep), but we have the opportunity to have them do it differently for our next experiments.
Thanks!!