Takara ssDNA-Seq Kit, 24 Rxns

The ssDNA-Seq Kit enables the construction of DNA libraries from both ssDNA and dsDNA and is ideal for samples that often contain a mixture of both DNA species, including FFPE DNA, DNA from environmental sources, and damaged or degraded DNA samples. This product contains reagents for 24 reactions.

ssDNA-Seq Kit protocol overview.While many DNA library preparation kits are limited to double-stranded DNA (dsDNA), this kit offers the flexibility to prepare high-quality sequencing libraries from both single-stranded (ssDNA) and double-stranded (dsDNA) inputs, producing high-quality libraries compatible with Illumina sequencers. Using single-strand DNA ligase (SDL) allows a stable library yield to be obtained from ssDNA that would be lost in conventional NGS library preparation of dsDNA, dsDNA that has become damaged or degraded, and even DNA that has undergone advanced fragmentation. Various DNA samples can be used as input, such as cell-free DNA (cfDNA), DNA derived from formalin-fixed paraffin-embedded (FFPE) specimens, ssDNA, combinations of ssDNA and dsDNA, environmental DNA, viral genome, short DNA, DNA aptamers, and synthetic oligos. Moreover, analysis is possible using trace amounts of DNA (10 pg) or ultra-short ssDNA (50 bases). The library preparation process consists of the five steps shown in this figure.

Confidently capture ssDNA and dsDNA with the ssDNA-Seq Kit.DNA libraries were prepared with the ssDNA-Seq Kit or ThruPLEX® Tag-Seq FLEX using a mixture of synthetic ssDNA and dsDNA. Paired-end sequencing (2 x 150 bp) was performed on the NextSeq® 2000 instrument. Bowtie2 was used for analysis and aligned to each known sequence. It was shown that the ThruPLEX Tag-Seq FLEX can capture dsDNA, while the ssDNA-Seq Kit can capture both ssDNA and dsDNA.

High-quality libraries prepared from human gDNA and FFPE samples.DNA libraries were prepared using the ssDNA-Seq Kit from human genomic DNA and FFPE-derived DNA samples with varying degrees of fragmentation. Input DNA, 10 ng for each sample, was fragmented using an ultrasonic generator (Covaris). Evaluation of the prepared libraries with the TapeStation HD5000 (Agilent Technologies) confirmed expected DNA library traces across all samples, regardless of their initial fragmentation level. Paired-end sequencing (2 x 150 bp) on the NextSeq® 2000 instrument, followed by alignment to GRCh38 using Bowtie2, demonstrated high mapping rates of over 97% from samples of any fragmentation level. DIN = DNA integrity number.

Successful generation of high-quality sequencing library from 50-base ssDNA oligos.DNA libraries were prepared using the ssDNA-Seq Kit from 10 ng of 160-base ssDNA synthetic oligos and 100 ng of 50-base synthetic oligos. Evaluation of the prepared libraries with the TapeStation (Agilent) confirmed good DNA library traces for each sample. The peak on the polymer side of the 50-base synthetic oligos is likely due to a bubble structure. Paired-end sequencing (2 x 150 bp) on the NextSeq® 2000 instrument and alignment to known 160-base and 50-base sequences using BWA-MEM confirmed mapping rates of over 97% for the 160-base synthetic oligos and over 89% for the 50-base synthetic oligos.

Uniform coverage across a wide range of GC content.DNA libraries were prepared using the ssDNA-Seq Kit from 10 ng (n = 2) of human genomic DNA fragmented with an ultrasonic generator (Covaris). Paired-end sequencing (2 x 150 bp) was performed on the NextSeq® 2000 instrument, with reads downsampled to a total of 9 million reads. The vertical gray bars represent the expected GC content distribution using 100 bp windows. These results confirm that the ssDNA-Seq Kit exhibits uniform coverage regardless of GC content.

Highest library yield from FFPE samples as compared to competitor kits. Panel A.DNA derived from FFPE samples with different degradation degrees was validated using the TapeStation Genomic DNA (Agilent) and the Takara FFPE DNA QC All-in-One Kit (Cat. # NN0001). Lane m: marker. Lane 1: FFPE colon (normal). Lane 2: FFPE adipose tissue (normal). Lane 3: FFPE lymph nodes (normal). Lane 4: FFPE kidney (tumor).Panel B.The ssDNA-Seq Kit had the highest library yield among the companies for all FFPE-derived DNA. DIN = DNA integrity number.

Highest mapping rate and low duplicate rate as compared to competitor kits. Panel A.The mapping rate of Competitor A, B, and C kits decreased significantly as DNA degradation progressed. On the other hand, ssDNA-Seq Kit showed a high mapping rate of over 97% and a low duplicate rate (Panel B) for all FFPE sample-derived DNA, regardless of the degradation degree. ssDNA-Seq Kit produces libraries regardless of the DNA degradation degree and obtains high-quality sequencing results.

cfDNA library coverage uniformity as compared to competitor kits.Libraries were prepared according to the protocol of each kit using 10 ng of cfDNA extracted from human plasma. Paired-end sequencing (2 x 150 bp) was performed on a NextSeq® 2000 instrument and reads were downsampled to a total of 4 million reads before analysis using Picard. The vertical gray bars represent the expected GC content distribution using 100 bp windows. The ssDNA-Seq Kit showed the most uniform coverage, independent of GC content. The ssDNA-Seq Kit, which provides high-quality sequencing results, is optimal for the analysis of cfDNA.