Technical data

5 Indexing and Sample Processing for Multiplexed Sequencing
Step 5. Pool samples for multiplexed sequencing
84 SureSelect
XT
Target Enrichment System for Illumina Multiplexed Sequencing
Step 5. Pool samples for multiplexed sequencing
The number of indexed libraries that may be multiplexed in a single
sequencing lane is determined by the output specifications of the platform
used, together with the amount of sequencing data required for your
research design. Calculate the number of indexes that can be combined
per lane, according to the capacity of your platform and the amount of
sequencing data required per sample.
1 Combine the libraries such that each index-tagged sample is present in
equimolar amounts in the pool. For each library, use the formula below
to determine the amount of indexed sample to use.
Volume of Index
Vf Cf
# Ci
---------------------------------
=
where V(f) is the final desired volume of the pool,
C(f) is the desired final concentration of all the DNA in the pool
# is the number of indexes, and
C(i) is the initial concentration of each indexed sample.
Table 45 shows an example of the amount of 4 index-tagged samples
(of different concentrations) and Low TE needed for a final volume of
20 µL at 10 nM.
2 Adjust the final volume of the pooled library to the desired final
concentration.
Table 45 Example of indexed sample volume calculation for total volume of 20 µL
Component V(f) C(i) C(f) # Volume to use (µL)
Sample 1 20 µL 20 nM 10 nM 4 2.5
Sample 2 20 µL 10 nM 10 nM 4 5
Sample 3 20 µL 17 nM 10 nM 4 2.9
Sample 4 20 µL 25 nM 10 nM 4 2
Low TE 7.6