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 Type | Core Rcmd. | Minimum Rqmt. | Sample Volume |
|
|---|---|---|---|---|
| (S-B) | (Bisulfite Seq) DNA Methylation Lib Prep | 1 ug | 200 ng | in 40 ul |
| (S-Ca) | Shotgun PE DNA Lib Prep, Gel-free | 2 ug | 50 ng | in 50 ul |
| (S-Cb) | Shotgun PE DNA Lib Prep, Gel-plus | 3 ug | 1 ug | in 50 ul |
| (S-D) | Shotgun LIPE DNA Lib Prep, Gel-plus | 3 ug | 1 ug | in 50 ul |
| (S-E) | Small RNA Lib Prep (miRNA) | 2 ug | 1 ug | in 5 ul |
| (S-G) | Degradome Lib Prep (PARE) | 100 ug | 75 ug | in 100 ul |
| (S-H) | Hi-C DNA Lib Prep | |||
| (S-I) | (ChIP-Seq) ChIP DNA Lib Prep | 100 ng | 10 ng | in 50 ul |
| (S-Ja) | (DAP-Seq) gDNA Lib Prep for TF Binding | 3 ug | 1 ug | in 50 ul |
| (S-Jb) | (DAP-Seq) Indexing PCR | 10 ng | 1 ng | in 20 ul |
| (S-L) | Ready-to-Seq Library | 100 ng | 25 ng | in 10 ul |
| (S-Ma) | MP DNA Lib Prep, Gel-free | 2 ug | 1 ug | in 50 ul |
| (S-Mb) | MP DNA Lib Prep, Gel-plus (4 sizes) | 10 ug | 6 ug | in 300 ul |
| (S-Mc) | MP DNA Lib Prep, Gel-plus (6 sizes) | 12 ug | 12 ug | in 300 ul |
| (S-P) | Nextera Indexing PCR (2nd Step PCR) | 100 ng | 10 ng | in 10 ul |
| (S-R) | Nextera PE DNA Lib Prep | 1 ug | 50 ng | in 30 ul |
| (S-Sa) | Stranded RNA Lib Prep, Poly-A | 3 ug | 100 ng | in 50 ul |
| (S-Sb) | Low-input Strnd RNA Lib Prep, Poly-A | 100 ng | 10 ng | in 50 ul |
| (S-Ta) | Stranded RNA Lib Prep, Ribo-zero | 3 ug | 1 ug | in 30 ul |
| (S-Tb) | Low-input Strnd RNA Lib Prep, Ribo-zero | 100 ng | 10 ng | in 10 ul |
| (S-X) | Human Whole Exome Capture | 1 ug | 50 ng | in 50 ul |
| (S-Y) | Target Panel Capture (Customized Prep) | 1 ug | 50 ng | in 50 ul |
| (X-VTO) (X-VGE) | 10X Visium Tissue Optimization 10X Visium Gene Expression | visit website |
Quantity Requirement for PacBio Sample
| Application Type | Core Rcmd. | Minimum Rqmt. | Sample Volume | Majority of DNA |
|
|---|---|---|---|---|---|
| (N-D) | ONT gDNA Lib Prep, Regular 30K | 6 ug | 3 ug | in 100 ul | > 50KB |
| (N-D) | ONT gDNA Lib Prep, Long 50K | 10 ug | 5 ug | in 100 ul | > 100KB |
| (P-Aa) | Amplicon PCR Barcoding | 10 ng | 2 ng | in 20 ul | |
| (P-Ab) | Amplicon Lib Prep | 1 ug (pooled) | 500 ng (pooled) | in 50 ul | |
| (P-D) | Low-input DNA Lib Prep | 2 ug | 200 ng | in 100 ul | > 20KB |
| (P-F) | Full-length 16S Amplification | 50 ng | 5 ng | in 20 ul | |
| (P-Ga) | gDNA Lib Prep, Regular 10KB | 6 ug | 6 ug | in 100 ul | > 30KB |
| (P-Gb) | gDNA Lib Prep, Long 20KB | 10 ug | 5 ug | in 100 ul | > 50KB |
| (P-H) | HiFi Lib Prep | 30 ug | 15 ug | in 100 ul | > 40KB |
| (P-I) | RNA Iso-Seq Lib Prep | 3 ug | 1 ug | in 5 ul | |
| (P-M) | Multiplexed Microbial Lib Prep | 2 ug | 1 ug | in 100 ul | > 30KB |
| (P-R) | Low-input RNA Iso-Seq Lib Prep | 500 ng | 500 ng | in 10 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) and Fragment Analyzer (Agilent; visit website) provide a visual assessment with an electropherogram and gel-like image to describe the intactness of samples. The rRNA ratios and RIN (or RQN) values are used to infer RNA integrity.
| Genomic DNA | OD260/280 = 1.8~1.9; OD260/230 ≥ 1.8 Qubit/Nanodrop ratio = 0.8~1.0 0.6% gel analysis (1X TAE; post-run staining): intact chromosome band above 23 kb and no degradation/smear observed RNase treated (< 10% RNA out of DNA) 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 | OD260/280 ≥ 1.9; OD260/230 ≥ 1.8 2% gel analysis (1X TAE; post-run staining): clear rRNA bands (28S:18S ~ 2:1) DNase treated (< 10% DNA out of total RNA) BioA/FA traces (recommended): rRNA ratio (28S/18S) ≥ 1.8; RIN value ≥ 8 |
| Amplicon | OD260/280 = 1.8~1.9; OD260/230 ≥ 1.8 2% gel analysis (1X TAE; post-run staining) BioA/FA traces (recommended): size range at 200-700 bp |
| ds cDNA | OD260/280 = 1.8~1.9; OD260/230 ≥ 1.8 BioA/FA traces (recommended) |
| ChIP DNA | OD260/280 = 1.8~1.9; OD260/230 ≥ 1.8 BioA/FA traces (recommended): size range at 100-500 bp after shearing; major at ~250 bp qPCR with gene-specific primers (recommended) |
| Ready-to-seq Library | OD260/280 = 1.8~1.9; OD260/230 ≥ 1.8 BioA/FA traces (recommended) qPCR result (recommended) |
