NextGen DNA Sequencing I Sample Requirements

ICBR-NextGenSeq@ad.ufl.edu   ///  (352) 273-8050   ///    RRID:SCR_019152

SAMPLE REQUIREMENTS

Please review our sample requirements before proceeding 

10X Genomics Sample Requirements

Illumina Sample Requirements

Illumina Customer-constructed Library Requirements

PacBio Sample Requirements

Oxford NanoPore

For DNA:

  1. Purity: Abs 260/280= 1.8-2.0 and Abs 260/230> 1.5 as determined in the NanoDrop or equivalent.
  2. Integrity: Fragments should be > 20 kb

 

For RNA:

  1. Purity: Abs 260/280= 1.8-2.0 and Abs 260/230> 1.5 as determined in the NanoDrop or equivalent.
  2. RNA Integrity Number (RIN) must be 7or higher

 

 

Comparison of minimum sample for library construction between Oxford NanoPore (ONT) and PacBio (PB)
Application NanoPore PacBio Comments
Large-insert genomic DNA without barcoding Smaller amounts if library is to be sequenced in 1-2 runs (1-5 µg) Larger amounts. However, the library yield is typically good for many runs (2-20 µg) Sample input mass for library prep varies depending on the desired insert size. ONT has no upper limits for input genomic fragment size. Ligation and loading are much faster for ONT
Large-insert genomic DNA with barcoding Similar amounts if library is to be sequenced in 2-3 runs (1-5 µg/sample) Similar amounts. However, the library yield is typically good for many runs (1-10.0 µg/sample) Sample input mass for library prep varies depending on the desired insert size. PB has an upper limit of 15-20 kb for proper demultiplexing. No size limit for ONT. Ligation and loading are much faster for ONT
RNAseq, cDNA with Amp 50 ng total RNA or 1-2 ng mRNA 300 ng Initial steps of protocols are very similar. However, the adaptor ligation and loading steps are much faster for ONT
Direct RNAseq 500 ng poly-A plus RNA (mRNA) N/A Sequencing output is ~1/10th in number of reads versus cDNA with Amp