Sample Requirement


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 TypeCore
(S-B)(Bisulfite Seq) DNA Methylation Lib Prep1 ug200 ngin 40 ul
(S-Ca)Shotgun PE DNA Lib Prep, Gel-free2 ug50 ngin 50 ul
(S-Cb)Shotgun PE DNA Lib Prep, Gel-plus3 ug1 ugin 50 ul
(S-D)Shotgun LIPE DNA Lib Prep, Gel-plus3 ug1 ugin 50 ul
(S-E)Small RNA Lib Prep (miRNA)2 ug1 ugin 5 ul
(S-G)Degradome Lib Prep (PARE)100 ug75 ugin 100 ul
(S-H)Hi-C DNA Lib Prep
(S-I)(ChIP-Seq) ChIP DNA Lib Prep100 ng10 ngin 50 ul
(S-Ja)(DAP-Seq) gDNA Lib Prep for TF Binding3 ug1 ugin 50 ul
(S-Jb)(DAP-Seq) Indexing PCR10 ng1 ngin 20 ul
(S-L)Ready-to-Seq Library100 ng25 ngin 10 ul
(S-Ma)MP DNA Lib Prep, Gel-free2 ug1 ugin 50 ul
(S-Mb)MP DNA Lib Prep, Gel-plus (4 sizes)10 ug6 ugin 300 ul
(S-Mc)MP DNA Lib Prep, Gel-plus (6 sizes)12 ug12 ugin 300 ul
(S-P)Nextera Indexing PCR (2nd Step PCR)100 ng10 ngin 10 ul
(S-R)Nextera PE DNA Lib Prep1 ug50 ngin 30 ul
(S-Sa)Stranded RNA Lib Prep, Poly-A3 ug100 ngin 50 ul
(S-Sb)Low-input Strnd RNA Lib Prep, Poly-A100 ng10 ngin 50 ul
(S-Ta)Stranded RNA Lib Prep, Ribo-zero3 ug1 ugin 30 ul
(S-Tb)Low-input Strnd RNA Lib Prep, Ribo-zero100 ng10 ngin 10 ul
(S-X)Human Whole Exome Capture1 ug50 ngin 50 ul
(S-Y)Target Panel Capture (Customized Prep)1 ug50 ngin 50 ul
10X Visium Tissue Optimization
10X Visium Gene Expression
visit website

Quantity Requirement for PacBio Sample

Application TypeCore
of DNA
(N-D)ONT gDNA Lib Prep, Regular 30K6 ug3 ugin 100 ul> 50KB
(N-D)ONT gDNA Lib Prep, Long 50K10 ug5 ugin 100 ul> 100KB
(P-Aa)Amplicon PCR Barcoding10 ng2 ngin 20 ul
(P-Ab)Amplicon Lib Prep1 ug (pooled)500 ng (pooled)in 50 ul
(P-D)Low-input DNA Lib Prep2 ug200 ngin 100 ul> 20KB
(P-F)Full-length 16S Amplification50 ng5 ngin 20 ul
(P-Ga)gDNA Lib Prep, Regular 10KB6 ug6 ugin 100 ul> 30KB
(P-Gb)gDNA Lib Prep, Long 20KB10 ug5 ugin 100 ul> 50KB
(P-H)HiFi Lib Prep30 ug15 ugin 100 ul> 40KB
(P-I)RNA Iso-Seq Lib Prep3 ug1 ugin 5 ul
(P-M)Multiplexed Microbial Lib Prep2 ug1 ugin 100 ul> 30KB
(P-R)Low-input RNA Iso-Seq Lib Prep500 ng500 ngin 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 DNAOD260/280 = 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 RNAOD260/280 ≥ 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
AmpliconOD260/280 = 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 cDNAOD260/280 = 1.8~1.9; OD260/230 ≥ 1.8

BioA/FA traces (recommended)
ChIP DNAOD260/280 = 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 LibraryOD260/280 = 1.8~1.9; OD260/230 ≥ 1.8

BioA/FA traces (recommended)

qPCR result (recommended)