QIAseq Ultralow Input Library Kits start with double-stranded DNA that has been fragmented enzymatically, chemically, mechanically or naturally. Kits use an optimized end-polishing reaction and a new Ultralow Input Ligation formulation, along with QIAGEN’s proprietary HiFi PCR Master Mix. This combination maximizes the conversion rate of sample DNA into the NGS library, while efficiently and evenly amplifying even high and low GC content regions of the genome. This protocol enables the highest possible yield sequencing library, free of adapter dimer contamination – starting from a little as 10−100 pg* of DNA input. Due to the kit’s flexible protocol, the same kit can also be used for higher DNA input amounts, including PCR-free library preparations from as little as 10 pg or as high as 100 ng of DNA input.
*Note that the genomic complexity necessary for a given experiment will vary depending on the genome size of the organism and the fraction of that genome included in the target region of interest.
Following adapter ligation and library amplification steps, reaction cleanup and removal of residual adapter dimers can be achieved by using Agencourt AMPure XP beads – which enable easy automation on various high throughput automation platforms.
Optional amplification of library DNA
PCR-based library amplification is normally required if the input DNA amount is below 100 ng. This protocol is optional, and enables high-fidelity amplification of the DNA library using the QIAseq HiFi PCR Master Mix that is included in the kit.
Contents and accessories
Dual-barcoded, plate-format adapters are included with the 96-reaction size QIAseq Ultralow Input Library Kit. Each well in the 96-plex adapter plate contains a single-use adapter consisting of a unique combination of two eight-nucleotide identification barcodes. By combining one i5 barcode and one i7 barcode in each ready-to-use adapter, the 96 reaction QIAseq Ultralow Input Kits supports up to 96-plex pooling of libraries prior to sequencing.
While adapters are not included in the 12-reaction size kit, two sets of 12-plex adapters with a single six-nucleotide barcode can each be ordered separately (QIAGEN cat. nos. 180985, 180986).