Sample Requirement

Quantification

The accurate input of nucleic acid for successful library preparation relies on the precise quantification.

Spectrophotometer (e.g. NanoDrop) detects all matters which absorbs light per given wave length, so both DNA and RNA are detected at OD260, and may lead to over estimation of concentration, especially for DNA. All gDNA samples are recommended to submit at 2-3X higher than required input amount due to frequent issues of concentration over estimation by spectrophotometer.

The fluorometric method is recommended to determine the amount of nucleic acid. In NGS core lab, we use Qubit Assays (Invitrogen; visit website) to determine the specific concentration per nucleic acid type, and decide whether the sample reaches the requirement of the input amount.

Quantity Requirement for Illumina Sample

 Application TypeRecommend QuantityVolume & Concentration
(S-B)Methylation DNA (Bisulfite Seq)1 – 2 ugin 25 ul
(≥ 40 ng/ul)
(S-C)Paired-End DNA2 – 3 ugin 50 ul
(≥ 40 ng/ul)
(S-D)Long Insert PE DNA2 – 3 ugin 50 ul
(≥ 40 ng/ul)
(S-E)Small RNA (miRNA)8 – 10 ugin 10 ul
(≥ 200 ng/ul)
(S-G)Degradome100 – 150 ugin 100 ul
(≥ 1000 ng/ul)
(S-I)ChIP DNA (ChIP Seq)5 – 10 ngin 25 ul
(≥ 0.2 ng/ul)
(S-L)Ready-to-Seq Libraryin 20 ul
(≥ 10 nM)
(S-M)Mate-Pair DNA10 – 20 ugin 100 ul
(≥ 100 ng/ul)
(S-N)Synthetic Long-Read DNA1 – 2 ugin 50 ul
(≥ 20 ng/ul)
(S-P)Indexing PCR (2nd Step PCR)≥ 300 ngin 20 ul
(≥ 15 ng/ul)
(S-Q)Nextera DNA75 – 150 ngin 25 ul
(≥ 3 ng/ul)
(S-R)Nextera XT DNA2 – 5 ngin 10 ul
(≥ 0.2 ng/ul)
(S-S)Stranded RNA, Poly-A4 – 6 ugin 50 ul
(≥ 80 ng/ul)
(S-T)Stranded RNA, Ribo-zero4 – 6 ugin 50 ul
(≥ 80 ng/ul)
(S-X)Human Exome Capture2 – 3 ugin 50 ul
(≥ 40 ng/ul)

Quantity Requirement for PacBio Sample

 Application TypeRecommend QuantityVolume & Concentration
(P-A)Full Length Amplicon Sequencing5 – 10 ugin 50 ul
(≥ 100 ng/ul)
(P-G)Genome Sequencing30 – 60 ugin 200 ul
(≥ 150 ng/ul)
(P-I)Isoform Sequencing (Iso-Seq)3 – 10 ugin 10 ul
(≥ 300 ng/ul)

 

Quality Assessment

For successful library construction, it is important to use high-quality nucleic acid as the starting material.

Absorbance ratios OD260/280 and OD260/230 are used to indicate the sample purity. Generally both ratios are around 1.8-1.9 for pure DNA, and 1.9-2.0 for pure RNA.

Agarose gel analysis is required to assess the sample integrity and potential cross-contamination between DNA/RNA. TAE buffer is recommended for electrophoresis, and gels should be run at maximum 8V per cm of gel length. Gel percentage should be adjusted depending on the sample type (0.6-0.8% for gDNA; 1.5-2% for RNA and amplicons <1.5kb). Molecular weight markers should cover 100bp~12kb range (λ-HindIII Ladder or Invitrogen 1KB Extension DNA Ladder is highly recommended) . All gels should be stained only AFTER electrophoresis in order to have even staining across the whole range.

Bioanalyzer 2100 (Agilent; visit website) provides a visual assessment with an electropherogram and gel-like image to describe the intactness of samples. The rRNA ratios and RIN values are used to infer RNA integrity.

Genomic DNA
(1) OD260/280 = 1.8~1.9; OD260/230 ≥ 1.8
(2) Qubit/Nanodrop ratio = 0.8~1.0
(3) 0.6% gel analysis (1X TAE; post-run staining): intact chromosome band above 23 kb and no degradation/smear observed
(4) RNase treated (< 10% RNA out of DNA)
(5) DNase I digestion test: treat 0.5 ug of gDNA with 0.1U of DNase I (25˚C for 5 min and 95˚C for 5 min), and check the digestion pattern on 3% agarose gel (1X TAE; post-run staining)

Total RNA
(1) OD260/280 ≥ 1.9; OD260/230 ≥ 1.8
(2) 2% gel analysis (1X TAE; post-run staining): clear rRNA bands (28S:18S ~ 2:1)
(3) DNase treated (< 10% DNA out of total RNA)
(4) BioAnalyzer traces: rRNA ratio (28S/18S) ≥ 1.8; RIN value ≥ 8

Amplicon
(1) OD260/280 = 1.8~1.9; OD260/230 ≥ 1.8
(2) 2% gel analysis (1X TAE; post-run staining)
(3) BioAnalyzer traces: size range 200-700 bp

ds cDNA
(1) OD260/280 = 1.8~1.9; OD260/230 ≥ 1.8
(2) BioAnalyzer traces

ChIP DNA
(1) OD260/280 = 1.8~1.9; OD260/230 ≥ 1.8
(2) BioAnalyzer traces: size range 100-500 bp after shearing; major at ~250 bp
(3) qPCR with gene-specific primers (recommended)

Ready-to-seq Library
(1) OD260/280 = 1.8~1.9; OD260/230 ≥ 1.8
(2) BioAnalyzer traces
(3) qPCR result (recommended)