Input Requirements

Input Requirements

Type of DNA

Supported Amplicon Size (bp)

DNA Quality

△Cq

Input (ng)1, 2

Genomic DNA

150, 175, 250

High

N/A

10

FFPE genomic DNA

150, 175

High

-1 – 1

10

 

 

Medium

> 1.0 – 2.5

20 – 50

 

 

Low

2.5 – 4.0

50 – 100

1The input amount depends on the △Cq. For more information, see the TruSeq FFPE DNA Library Prep QC Reference Guide (document # 1000000002136).

2Greater input increases library yield and improves sequencing metrics such as specificity (percent aligned reads).

Input DNA Quantification

Quantify the starting genomic material using a fluorescence-based quantification method, such as a Qubit dsDNA Assay Kit or PicoGreen, rather than a UV-spectrometer-based method. Fluorescence-based methods, which employ a double-stranded DNA (dsDNA) specific dye, specifically and accurately quantify dsDNA even in the presence of many common contaminants. In contrast, UV spectrometer methods based on 260 OD readings are prone to overestimating DNA concentrations due to the presence of RNA and other contaminants commonly found in genomic DNA (gDNA) preparations.

Assessing DNA Quality

Use the TruSeq FFPE DNA Library Prep QC Kit (catalog # FC-121-9999) to determine FFPE DNA quality and input amounts for the TruSeq Custom Amplicon Low Input Library Prep Kit.

Input DNA Dilution

Quantify and dilute the DNA to the desired input amount based on the DNA type and quality. You can dilute and store more than the required DNA for use later. Dilute DNA in RS1 and SS1 and store as described in the Quantify and Dilute DNA section in the TruSeq Custom Amplicon Low Input Library Prep Reference Guide (document # 1000000002191).