Sample Guidelines

The key to successfully performing NGS is to provide us, the BIG Core, with source materials, DNA, RNA, and/or cells, of sufficient quality and quantity to produce a high-quality NGS library for sequencing. By far, the quality of the initial materials is the single most important factor in the whole process.

DNA

Any source of DNA, usually double-strand DNA (dsDNA), can be NGSed, including genomic DNA (gDNA), metagenomic DNA, BACs, PCR amplicons, ChIP enriched DNA, and those reverse transcribed from RNA. For library construction of Illumina short-read sequencing, the dsDNA is often fragmented into small sizes, usually not exceeding 600 bp in length, using either physical shear or enzymatic tagmentation. In contrast, for PacBio long-read sequencing, the dsDNA is required as intact as possible for the construction of an NGS library in large sizes, up to 30 kb. As such, extreme care should be taken in the purification of high molecular weight (HMW) DNA, as well as the following storage and/or shipment. Avoid vortexing wherever possible and use wide bore tips for pipetting.

A number of well-established DNA extraction commercial kits and protocols are available for a variety of samples. Given the tremendous variety, we do not give specific recommendations or perform DNA purification for our customers, unless special requests, such as batch process in a large scale with automation or HMW DNA purification, are raised. Investigators need to carefully choose from these DNA extraction methods for their specific needs.

The BIG Core requires high-quality DNA (260/280 UV absorbance ratio close to 1.8; 260/230 ratio >2.0) in elution buffer (EB, Tris-Cl 10 mM, pH 8.5) or nuclease-free water for NGS. Quantity requirement for DNA sample submission is listed as follows, which is based on fluorometric quantification method (e.g., Qubit), since quantity measurement by a spectrophotometer (e.g., NanoDrop) is unreliable, often over-assessed.

  • Illumina DNA Prep: 1 – 500 ng DNA for small genomes (e.g., microbial) and 100 – 500 ng for large genomes (e.g., human).
  • Illumina DNA Prep with Enrichment: 50 – 1000 ng gDNA.
  • ChIP DNA Prep: > 10 ng ChIP enriched DNA in 20 µl or less of water, in a low retention tube. The ChIP DNA should be mostly shorter than 500bp.
  • PacBio Standard HiFi Library Prep: > 5 µg HMW gDNA for an up to 3 Gb genome.
  • PacBio Low DNA Input Library Prep: > 500 ng HMW gDNA for a genome size limited at 1 Gb.
  • PacBio Ultra-Low DNA Input Library Prep: > 5 ng HMW DNA for a genome size limited at 500 Mb

 

RNA

RNA in high quality is essential for successful RNA-Seq experiments. A number of well-established RNA extraction commercial kits and protocols are available for a variety of species, cell/tissue types. Given the tremendous variety, we do not give specific recommendations or perform RNA purification for our customers. Investigators need to carefully choose from these methods for RNA extraction from their specific species, tissues, and cells, as these methods directly impact the quality and quantity of RNA obtained. If possible, total RNA purification should immediately follow tissue/cell isolation to prevent RNA degradation and/or alterations in the transcript profile. If immediate RNA purification is not possible, cells/tissues must be stored with proper RNA preservatives, such as RNAlater, DNA/RNA shield, and/or Monarch DNA/RNA Protection Reagent.

Notably, RNA is often contaminated by DNA, though the 260/280 ratio determined by a spectrophotometer in RNA purity measurement is over 1.8. DNA contamination may interfere with RNA-Seq. If investigators use column-based purification methods, such as RNeasy and Direct-Zol, for RNA extraction, on-column DNase I digestion during RNA purification is highly recommended to reduce DNA contamination.

Changes in gene expression can be attributed to a variety of factors, including the person performing the experiment, the materials started with, the methods employed in the process, and the intracellular regulatory genes potentially altered. To control for various sources of variability, a common set of external RNA controls has been developed by the External RNA Controls Consortium (ERCC), an ad-hoc group of academic, private, and public organizations hosted by the National Institute of Standards and Technology (NIST). The ERCC Spike-In Control Mixes consist of a set of synthetic, unlabeled, polyadenylated transcripts with different lengths, which mimic natural eukaryotic mRNAs. We highly recommend the ERCC Spike-In Control Mix(es) being added as early as possible to an RNA-Seq experiment in order to increase the gene expression analysis accuracy and to evaluate the whole analysis sensitivity. Please let the BIG Core be advised if the RNA samples contain the ERCC Spike-In Control.

We strongly encourage pilot projects to confirm that the chosen methods will reproducibly yield the required amount of high-quality RNA from certain cells/tissues. Once an RNA extraction protocol is established, it is important that all the samples collected for a given project are treated with the same protocol. Changes in these techniques may result in differences in the transcript profile, which may be misidentified as changes due to experimental treatment, while in reality, they are artifacts of varying extraction and storage methodology.

RNA samples should be stored in a -80°C freezer and shipped on dry ice. RNA samples received at the BIG Core for RNA-Seq will be quantity measured by Qubit 4 fluorometer and quality checked with Agilent 4200 TapeStation. Please be aware, measurement by a spectrophotometer (e.g., NanoDrop) often over-estimates the quantity of material, whereas a fluorometer (e.g., Qubit) detects fluorescent dye specifically binding to RNA and enables more accurate assessment. The TapeStation will produce an RNA Integrity Number (RIN), an objective measure of RNA quality based on prokaryotic 23S/16S or eukaryotic 28S/18S rRNA ratio. RIN scores vary from 1-10, with 10 being the highest quality sample showing the least degradation.

The BIG Core requires a minimum of 500 ng of total RNA with RIN > 7 in 50 µL or less RNase-free water for RNA-Seq library preparation. For small RNA (microRNA) sequencing, RNA in higher concentration is preferred; for PacBio full-length RNA-Seq, i.e., Iso-Seq, RNA in higher RIN is preferred; for RNA-Seq with rRNA depletion, such as formalin-fixed paraffin-embedded (FFPE) samples, DV200 > 50% is preferred. Investigators will be consulted if any sample does not meet these requirements, before RNA-Seq library construction.

Cells

A suspension of viable single cells is critical to obtaining high-quality single-cell sequencing data. It is important to minimize the presence of cellular aggregates, dead cells, cells of no interest, cell debris, non-cellular nucleic acids, and potential polymerase inhibitors in the single-cell suspension. Notably, during the procedure of single-cell sequencing analysis, dead cells and cell debris may release excess DNA/RNA, cause other cells to clump, and clog the channels of microfluidic chips, which could lead to single-cell capture failure. Therefore, avoid vortexing wherever possible and use wide bore tips for pipetting of single cells.

A difficulty in single-cell sequencing analysis is that the cells of interest may be a very small part of the population in the tissue. Although new technologies now allow the sequencing of thousands of cells, the low frequency of cells of interest may still affect the analysis cost and efficiency. Cell population enrichment can thus be used with MACS (magnetic-activated cell sorting) or FACS (fluorescence-activated cell sorting). Both MACS and FACS are antibody-based isolation methods targeting cell surface antigen(s) or biomarker(s). In comparison, MACS is much faster but much less versatile, than FACS. Given the variety and complexity of antibodies, we do not perform MACS or FACS for our customers. For details of FACS, please contact ECU Flow Cytometry Core.

To provide high-quality single-cell sequencing, we offer investigators access to Miltenyi Biotec GentleMACS Octo tissue dissociator with heaters. By appointment, investigators may utilize the instrument to develop fully automated protocols for dissociating various tissues of interest into cell suspensions, with or without MACS enrichment of cells of interest, ready for downstream applications. For optimal performance of single-cell suspension compatible with the 10× Genomics GemCode platform, please refer to the Single Cell Protocols – Cell Preparation Guide.

For single-cell sequencing analysis at the BIG Core, cells should be resuspended in PBS (without Mg2+, Ca2+, or EDTA) + BSA (up to 1%) at a density of more than 1×106 cells/mL with viability ideally greater than 90% and a minimum volume of 150 µL. Cells received will be quality checked with Countess 3 FL automated cell counter for cell density and viability. Investigators will be consulted if any cell sample does not meet the requirements, before any NGS library construction.

Libraries

The BIG Core accepts pre-made NGS libraries for sequencing. Especially, for 16S/ITS metagenomics sequencing, we encourage investigators to prepare the NGS libraries by themselves, so as to reduce any potential cross-contaminations. It is highly recommended the pre-made NGS libraries being quantity measured by Qubit 4 fluorometer and quality checked with Agilent 4200 TapeStation, before pooling any multiplexed libraries. The BIG Core requires a minimum of 20 µL of > 10 nM pre-made and pooled library in water or EB, provided in a low retention tube, as well as a sample sheet listing all the libraries and their individual index(es)/barcode(s) being used. Sequencing parameters and specifications can be discussed, such as specific custom sequencing primers needed. We will provide troubleshooting assistance, if needed, to ensure the successful completion of your project.

Storage

Due to space limitations at the BIG Core, samples/libraries will be stored for only three months after sequencing is completed. We appreciate that investigators pick up their samples/libraries in time after sequencing is complete, and we are happy to accommodate.