Double sided size selection and bead clean up

Some library preparation kits, such as Illumina DNA Prep and TruSeq DNA PCR Free, use a double-sided bead clean-up that removes both very small and very large library fragments. A double-sided clean-up is designed to narrow the library size range, leading to more consistent performance between libraries.

The first step, also referred to as the right-side clean-up, removes large library fragments. The large fragments are bound to the beads and left behind, while the library of interest remains in the supernatant and is transferred to a new well. New beads are added to the supernatant for the second clean-up, or the left-side clean-up. The desired library is now bound to the beads and must be washed and eluted after the supernatant, containing small library fragments and other unwanted small products, is removed.

Ratios for each step of a double-sided clean-up vary based on the desired final library size, and, as a rule, the right-side ratio is smaller than the left-side ratio. The calculation for the beads to library ratio for the right-side clean-up is the same as a single-sided clean-up, however the ratio calculation for the left-side clean-up accounts for the beads that were added in the first step as well as the volume of supernatant transferred from the right-side clean-up. The following equation is used to calculate the left-side clean-up ratio:

where Vo = Total volume of DNA sample + beads from first step

and Vt = Volume of supernatant transferred from the right-side clean-up.

For the calculation of the amount of beads added in the second step, a scale factor (Vo/Vt) is required as 100% of the supernatant cannot be transferred to a new well for the left-side clean-up. For example, in the TruSeq Nano DNA protocol only 250 µl of supernatant from a total volume of 260 µl (DNA sample + beads from the first step) is transferred from the right-side clean-up. In this case Vt = 250 µl and Vo = 260 µl, so the scale factor is Vo/Vt = 260/250 = 1.04.

Example calculation:

Double-sided size selection with a right-side clean-up ratio of 0.5x, a left-side clean-up ratio of 0.7x and an original DNA sample volume of 90 µl.

For the first step, 45 µl of beads are needed for a right-side clean-up ratio of 0.5x. Subsequently, 120 µl of supernatant are transferred from the right-side clean-to a new well. For the second step, a calculated amount of 16 µl beads is obtained from the formula based on a left-side clean-up ratio of 0.7x.

Vo = 90 µl + 45 µl = 135 µl; Vt = 120 µl

Figure 1: The red trace shows the final library after a double-sided size selection. In this case, the first step removes large fragments and the second step removes small fragments. More details on the double-sized selection can be found in the publication Bead-linked transposomes enable a normalization-free workflow for NGS library preparation.

Notes:

• Overall library yield is reduced with each bead clean-up.

• Check the specific library preparation protocol before starting the experiment to see if beads are included in the kit or must be purchased separately.

For any feedback or questions regarding this article (Illumina Knowledge Article #6157), contact Illumina Technical Support [e-mail].