Features

• Magnetic-bead based clean-up and size selection of NGS library preparation reactions
• Sample input as little as 17.5 pg
• Sample volume of 50 µL to 200 µL
• Tunable size selection 150-800 bp
• Convenient magnetic bead technology
• Easy to adjust for specific applications or sequencers
• Optimal scalability for manual and automated processing

Principle/Procedure

For NGS library preparation, reaction clean-up and fragment size selection is required for most sequencing platforms. NucleoMag® NGS Clean-up and Size Select enables clean-up reactions as well as a single or double side size selection to recover the fragment lengths which are needed in your specific application. The magnetic beads in the kit are already present in binding buffer and just have to be diluted to tune the required fragment size. The dilution ratio is similar to other beads in the market that existing recommendations in NGS library preparation protocols can be used and protocols do not have to be changed.

NucleoMag® NGS Clean-up and Size Select procedure

Size selection of fragment mix
For single side size selection (left or right), the sample is mixed with the beads in a certain ratio to exclude larger or smaller fragments until a chosen cut-off. For the double sized size selection two binding steps are performed, to exclude larger fragments above the cutoff and smaller fragments below the lower cutoff.

Application data

Recoveries of different fragment sizes

For DNA size selection 100 µL DNA (10 ng/µL) have been added to different volumes of NucleoMag® NGS Clean-up and Size Select beads to achieve the shown ratios (ratio = beads/sample). Input DNA contained fragment size from 100 bp to 1000 bp. The different recoveries of the used ratios (beads: input DNA) are shown in percentage [%].

Double size selection

Sheared DNA from mouse tissue has been double size selected by using different volume ratios.

green: relative size distribution from mouse tissue DNA after fragmentation
red: final library size distribution after double size selection (right 0.4//left 0.6)
(mean fragment size 460 bp)
blue: final library size distribution after double size selection (right 0.55// left 0.8)
(mean fragment size 340 bp)

Short fragment removal

Bioanalyzer traces and gel image of DNA ladder before and after clean-up using NucleoMag® NGS Beads and the protocol for removing adapter dimers.

Fragment size analysis of prepared NGS libraries

NGS libraries have been prepared using Truseq DNA PCR Free kit by Illumina (red), AMPure XP (blue) and NucleoMag® NGS Clean-up and Size Select (green) for clean-up and size selection steps. (A) Input DNA, 1 µg sheared E. coli DNA. (B) Size distribution of DNA Fragments after Library preparation as input for sequencing, the expected fragment size is 650 bp (insert+adapters).

A.        B.  

Calibration assay

The calibration assay has been developed to identify the optimal volume ratio of bead suspension to sample for your size selection procedure. 100 µL of DNA ladder (10 ng/µL) with fragments ranging from 50 bp to 1000 bp (REF) are mixed with different volumes of NucleoMag® NGS Bead Suspension. The ratios of bead suspension to sample used in the assay vary from 0.5 to 1.0, thus providing different fragment size cutoff parameters (ratio = beads/sample). Yields are determined by running the DNA eluted from the beads on an Agilent Bioanalyzer DNA 7500 LabChip.
 

Sequencing analysis

In order to compare the sequencing performance after library preparation generation with different clean-up and size selection products, the distribution of genome sequence coverage (depth of genome base coverage) and effect on GC content on sequence coverage have been tested. (A) No difference in mapped depth of genome base coverage. The depth is constant showing low bias. (B) Horizontally plot pattern. After the summary of aligned read bases within a 0.5 kb genome region, the two samples have been normalized/matched resulting in a horizontally pattern showing the similarity of the two samples.
NGS libraries have been prepared using Truseq DNA PCR Free kit by Illumina (red), AMPure XP (blue) and NucleoMag® NGS Clean-up and Size Select (green) for clean-up and size selection steps. Sequencing was performed on a HiSeq 2500 using TruSeq Rapid SBS Kit.

A. 

B.

Insert size in sequencing

After Sequencing target insert size was determined. Insert size was for all three library kits similar with a peak around 380 bp.