Implementation Best Practices: Sample Preparation

For DNA inputs between 100–500 ng, accurate quantification of the initial DNA sample is not required, and normalization of the final yield is expected.

If you are using less than 100 ng DNA input, we recommend quantification.  To quantify input genomic DNA and DNA in the PCR and enriched libraries, use a fluorometric-based method that is specific to double-stranded DNA, such as QuantiFluor or PicoGreen. The concentration of gDNA can be determined using the Qubit dsDNA BR Assay or the Qubit dsDNA HS assay. These assays use a fluorescent dye that is highly selective for double-stranded DNA over RNA and can detect samples in a concentration range from 10 pg/μl – 1000 ng/μl. PicoGreen dye can also be used to accurately measure the DNA concentration.

For more information, see the Genomic DNA Input Recommendations section of the Nextera DNA Flex Reference Guide.

The Nextera DNA Flex protocol is compatible with DNA inputs ranging from 1–500 ng. For human DNA samples and other large complex genomes, the recommended DNA input is between 100–500 ng. For small genomes, the DNA input amount can be reduced to as low as 1 ng (modifying the PCR cycling conditions accordingly). For normalized libraries, the recommendation DNA input is ≥ 100 ng.

For more information, see the Genomic DNA Input Recommendations section of the Nextera DNA Flex Reference Guide and the Nextera DNA Flex Question and Answers.

The minimum amount of input DNA without quantitating and utilizing the built-in normalization is ≥ 100 ng.

For more information, see the Genomic DNA Input Recommendations section of the Nextera DNA Flex Reference Guide.

Additional PCR cycles can be implemented. For more information, see the Genomic DNA Input Recommendations section of the Nextera DNA Flex Reference Guide.

Applicable sample types include:

• Small Genomes: 1–500 ng DNA; Large Genomes: 100–500 ng
• Blood
• Saliva
• Colonies
• Liquid Culture
• Stool Samples
• FFPE Samples

For instructions, see the Nextera DNA Flex Microbial Colony Extraction Protocol Guide.

Assess the quality of genomic DNA by running an aliquot of the sample (approximately 10–100 ng) on a 1% agarose gel stained with SYBR Stain. High quality, intact genomic DNA appears as a high molecular weight band (> 10,000 bp) in the absence of a lower molecular weight smear. Low molecular weight smearing can indicate the presence of RNA or degraded DNA. Avoid resuspending DNA In EDTA containing buffers such as TE (similar to Nextera DNA/Nextera XT). 

For more information, see the Assess DNA Quality section of the Nextera DNA Flex Reference Guide.

Nextera DNA Flex is optimized for gDNA (dsDNA) and will not work on ssDNA/RNA. 

Yes. The PCR amplicon must be > 300 bp. Shorter amplicons can be lost during the library cleanup step. Tagmentation cannot add an adapter directly to the distal end of a fragment, so a drop in sequencing coverage of ~50 bp from each distal end is expected. To ensure sufficient coverage of the amplicon target region, design primers to extend beyond the target region by 50 bp per end.  

Yes. A variety of genomes have been tested and the data remains consistent.  A variety of genomes prepared with Nextera DNA Flex (from bacteria, plants, agriculture, and human) can be found in BaseSpace Sequence Hub. 

Public Data in BaseSpace Sequence Hub does not yet have any iSeq Sequencing System runs, but is available for the following platforms:

• MiSeq Sequencing System: Nextera DNA Flex (replicates of E.coli, B.cereus, and R.sphaeroides)
• MiniSeq Sequencing System: Nextera DNA Flex (replicates of E.coli, B.cereus, and R.sphaeroides)

We recommend targeting a 260/230 ratio of 2.0– 2.2.  Values outside this range indicate the presence of contaminants that may cause incomplete tagmentation and adversely impact the final library yield.  

The ratio of absorbance at 260 nm to absorbance at 280 nm provides an indication of sample purity. This protocol is optimized for DNA with absorbance ratio values of 1.8–2.0, which indicates a pure DNA sample. Values outside this range indicate the presence of contaminants that may cause incomplete tagmentation and adversely impact the final library yield. For a complete list of contaminants, including sources, avoidance, and effects on the library, see the Nextera XT Troubleshooting Technical Note. Dilute the starting material in nuclease-free water.

NOTE: Incomplete tagmentation caused by contaminants may result in library preparation failure, poor clustering, or an unexpectedly high scaffold number.

For more information, see the Assess DNA Quality section of the Nextera DNA Flex Reference Guide.