NGS library prep from ssDNA—the ssDNA-Seq Kit

Prepare high-quality libraries from single-stranded DNA (ssDNA) species that are not captured during conventional double-stranded DNA (dsDNA) library preparation. 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.

Prepare high-quality libraries from single-stranded DNA (ssDNA) that are not captured during conventional double-stranded DNA (dsDNA) library preparation. 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.

The kit is compatible with DNA inputs as low as 10 pg and ssDNA fragments as short as 50 bp. Unlike traditional dsDNA-seq workflows, the ssDNA-seq Kit workflow begins with the conversion of all DNA species within the sample to ssDNA through a pretreatment and heat denaturation step. Following heat denaturation, Single-Strand DNA Ligase is used to add the first adaptor to the 3’ ends of all ssDNA fragments. The library is converted to dsDNA through primer extension, and the second adaptor is added to the 5’ ends of the dsDNA fragments. Finally, Unique Dual Indexes (Cat. # 634752–634756; sold separately) are used to add the indexing sequences needed for Illumina sequencing, and the library is amplified. This simple workflow can be completed in less than 2 hr.

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Cat. #	Product	Size	Price
NN0003	ssDNA-Seq Kit	24 Rxns	USD $1678.00
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. Notice to purchaser Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval. Documents Components Image Data Back ssDNA-Seq Kit protocol overview 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. Back Confidently capture ssDNA and dsDNA with the ssDNA-Seq Kit 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. Back High-quality libraries prepared from human gDNA and FFPE samples 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. Back Successful generation of high-quality sequencing library from 50-base ssDNA oligos 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. Back Uniform coverage across a wide range of GC content 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. Back Highest library yield from FFPE samples as compared to competitor kits 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. Back Highest mapping rate and low duplicate rate as compared to competitor kits 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. Back cfDNA library coverage uniformity as compared to competitor kits 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.
NN0004	ssDNA-Seq Kit	96 Rxns	USD $4335.00
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 96 reactions. Notice to purchaser Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval. Documents Components Image Data Back ssDNA-Seq Kit protocol overview 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. Back Confidently capture ssDNA and dsDNA with the ssDNA-Seq Kit 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. Back High-quality libraries prepared from human gDNA and FFPE samples 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. Back Successful generation of high-quality sequencing library from 50-base ssDNA oligos 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. Back Uniform coverage across a wide range of GC content 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. Back Highest library yield from FFPE samples as compared to competitor kits 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. Back Highest mapping rate and low duplicate rate as compared to competitor kits 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. Back cfDNA library coverage uniformity as compared to competitor kits 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.

Overview

Construct Illumina-ready NGS libraries from both ssDNA and dsDNA
Use with highly fragmented, damaged, or degraded DNA, including FFPE samples
Enable analysis of ssDNA fragments as short as 50 bp
Complete library prep in under 2 hr with a simple workflow
Multiplex up to 384 samples with Unique Dual Index Kit compatibility

The ssDNA-Seq Kit is compatible with many DNA forms not captured by traditional library prep kits

DNA form	Compatible with the ssDNA-Seq Kit?	Compatible with traditional dsDNA library prep kits?
dsDNA
ssDNA		Χ
dsDNA with nicks/gaps		Χ
dsDNA with 5’ overhangs		Χ
dsDNA with 3’ overhangs		Χ

More Information

Applications

Use in the analysis of:

ssDNA
dsDNA
Genomic DNA
Cell-free DNA
Ultra-short DNA
Synthetic oligos
FFPE DNA
Environmental DNA
Viral DNA
Ancient DNA
DNA aptamers

Additional product information

Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.

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NGS library prep from ssDNA—the ssDNA-Seq Kit

Notice to purchaser

ssDNA-Seq Kit protocol overview

Confidently capture ssDNA and dsDNA with the ssDNA-Seq Kit

High-quality libraries prepared from human gDNA and FFPE samples

Successful generation of high-quality sequencing library from 50-base ssDNA oligos

Uniform coverage across a wide range of GC content

Highest library yield from FFPE samples as compared to competitor kits

Highest mapping rate and low duplicate rate as compared to competitor kits

cfDNA library coverage uniformity as compared to competitor kits