xGen™ ssDNA & Low-Input DNA Library Preparation Kit

Product details

NGS DNA library construction from samples with damaged DNA

The xGen ssDNA & Low-Input DNA Library Prep Kit enables library preparation from damaged samples that can be difficult to obtain sequence information. The xGen ssDNA & Low-Input DNA Library Prep Kit can create libraries in 2 hours from as little as 10 pg of input material. Libraries can be prepared from heavily damaged samples by using IDT Adaptase technology. Unlike other library methods, the Adaptase technology generates library molecules from single-stranded DNA fragments, which allows better recovery of sample input DNA complexity from heavily nicked and degraded samples compared to other commercially available products for Illumina^® platforms. Sample types include degraded FFPE, ancient DNA, chromatin immunoprecipitation (ChIP), and other enriched samples that have undergone DNA damage.

Rescue valuable sequencing data from low-abundance samples with ssDNA content

The xGen ssDNA & Low-Input DNA Library Prep Kit is designed for users needing to sequence samples containing ssDNA for research. These include metagenomic and viromic samples where relative abundance of both ssDNA and dsDNA phage and viral genomes can be identified. Other compatible sample types include heat-denatured pathogenic samples, samples extracted under harsh denaturing conditions to decrease extraction bias, and oncology-related liquid biopsy samples when detection of single-stranded cfDNA content is desired.

Workflow

The workflow for this kit is shown in Figure 1. As illustrated in this diagram, samples undergo four steps:

1. Adaptase technology—the Adaptase reaction simultaneously performs tailing and ligation of R2 Stubby Adapter to the 3´ ends in a highly efficient, template-independent manner.
2. Extension—the extension reaction generates a second strand prior to ligation.
3. Ligation—ligation reaction adds R1 Stubby Adapter to the original strand.
4. Indexing PCR—PCR is used to incorporate sample indexes and sequences needed for Illumina^® sequencing. Indexing primers are supplied separately.

Specifications

Product data

Relative abundance of both ssDNA and dsDNA phage

The xGen ssDNA & Low-Input DNA Library Prep Kit was used to prepare and sequence three artificial viromes containing different proportions of the ssDNA phage PhiX174 and M13 mixed with dsDNA phage (Figure 2). In this experiment, all cases supported that the proportions were preserved when sequenced with the xGen ssDNA & Low-Input DNA Library Prep Kit without any prior whole genome amplification for identification of ssDNA phage. 

The libraries were sequenced on an Illumina^® MiSeq^® using v2 chemistry and 101 paired-end reads. Reads were then aligned with bowtie2 to assess the relative abundance of each virus, defined as the number of reads mapped normalized by genome size. These virome abundance values were then compared to the copy number of each genome in the AVC, estimated through SYBR^® Green counts for the dsDNA phages, or calculated based on the quantity of DNA measured with NanoDrop^® (Thermo Fisher) readings for ssDNA phages. The expected abundance of dsDNA phages was doubled to correct for the presence of two DNA molecules that can act as potential templates compared to a single potential template for ssDNA viruses.

DNA extraction and sequencing of a hard-to-extract microbe

The data in Figure 3A and B show results after DNA extraction and sequencing of Facklamia sp., a microbe recognized for the difficulty it presents for DNA extraction. Pure cultures of Facklamia sp. HGF4 were grown up to 2 x 10⁹ CFU/mL. Facklamia sp. HGF4 was selected because it is a Gram-positive isolate with gDNA that is difficult to extract using a nondenaturing bead-beating extraction method. Because Facklamia sp. HGF4 does not have a reference genome, de novo assembly of the sequencing output was required. Bacterial culture was split into two aliquots—one from which gDNA was isolated using bead beating, and one from which the gDNA was extracted by denaturing NaOH boiling. Briefly, 1 mL of 2 x 10⁹ CFU/ml bacteria was pelleted by centrifugation, the supernatant was removed, and cells were resuspended in 50 µL of sterile water. Then, 50 µL of 0.1 N NaOH was added to the bacterial suspension, and the sample was mixed by vortexing and then boiled at 95 ^°C for 15 minutes. The solution was then chilled on ice and neutralized by adding 8 µL of Tris-HCl, pH 7.0. The sample was centrifuged at 15,000 x g for two minutes to pellet the cell debris and any unlysed cells, and the supernatant was transferred to a new reaction tube. The isolated gDNA was precipitated with isopropanol to clean and concentrate the sample. DNA recovery was quantified by Qubit^® (Thermo Fisher) for dsDNA and NanoDrop for ssDNA. Libraries were prepared using the xGen ssDNA & Low-Input DNA Library Prep Kit. Facklamia sp. HGF4 was assembled de novo using MIRA 4.0 and annotated using the RAST server.