Some library preparation kits, such as the Illumina DNA Prep (formerly known as Nextera® DNA Flex) and TruSeq® DNA PCR Free, use a double-sided bead clean-up that removes both very small library fragments and very large 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 is removed, which contains small library fragments and adapter dimers.

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 1^st step

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

For calculation of the amount of beads added in the 2^nd step, a scale factor (Vo/Vt) is required when 100% of the supernatant is not or cannot be transferred to a new well for the left-side clean-up. For example, in the TruSeq Nano DNA LP WF only 250 ul of supernatant from a total volume of 260 ul (DNA sample + beads from 1^st step) is transferred from the right-side clean-up. In this case Vt = 250 ul and Vo = 260 ul, 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 ul.

For the 1^st step, 45 ul of beads are needed for a right-side clean-up ratio of 0.5x. Subsequently, 120 ul of supernatant are transferred from the right-side clean-to a new well. For the 2^nd step, a calculated amount of 16 ul beads is obtained from the formula based on a left-side clean-up ratio of 0.7x.

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

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.