DNA samples for long-read sequencing library preparations or also 10X genomics linked-reads have to be exceptionally pure. Please see the sample requirements. For difficult DNA samples, especially all plant DNA samples with hard-to-remove contaminants (e.g.
Many types of RNA-seq require RNA samples of high integrity and high chemical purity - please see the sample requirements. If the tissue or cell samples are handled correctly (e.g.
We currently offer restriction-enzyme based reduced representation sequencing with ApeKI, MspI, NlaIII, PstI and SbfI enzymes, as well as a strictly PCR-based reduced representation sequencing protocol.
At the moment we only carry out high-molecular-weight DNA (HMW-DNA) isolations for the purpose of 10X Genomics and Nanopore sequencing. Please inquire with Ruta Sahasrabudhe, PhD.
We do not offer DNA isolations for Illumina sequencing and RNA isolations at the moment. However the Taqman Core does.
The best option for sequencing larger numbers of amplicons that exceed the MiSeq read length limitations (e.g. amplicons longer than 600 nt) is Pacbio sequencing.
Amplicons up to 12 kb length can generate highest quality sequence data for individual molecules employing circular-consensus-sequencing (CCS) analysis. Amplicons longer than 3.5 kb should be run with 20 hour movies (sequencing run times).
The isolation of high-quality DNA and RNA samples from plants can be challenging due to the presence of inhibiting and damaging phytochemicals. Thus, it is not possible to recommend a single protocol that works for all samples. In any case the RNA samples should be DNAse treated, and QC-ed on a Bioanalyzer for sample integrity and via Nanodrop for purity. Please see sample requirements.
For many species and
Bead based sample cleanups (e.g., Ampure XP, RNAClean XP) and spin column-based protocols (e.g., Qiagen, Zymo, NorgenBiotek) tend to be the most efficient ways to remove chemical contaminants. For genomic DNA samples to be sequenced on Illumina sequencers, we suggest spin columns since DNA treated this way will always dissolve well and completely.
Quality and quantity of DNA and RNA is critical for high quality sequencing output. Please make sure your DNA is not degraded and is free of RNA contamination. RNA samples should always be assessed on the bioanalyzer for the absence of gDNA contamination (can be removed with DNaseI treatment followed by a column clean-up; e.g. Zymo "RNA Clean and Concentrator") and degradation. Preferentially determine the concentrations of your DNA and RNA samples using fluorometry (e.g. with a Qubit or plate reader). The sample purity should be assessed by spectrophotometry (e.g.
