FREQUENTLY ASKED QUESTIONS
ACGT has significant experience in preparing various libraries, both RNA and DNA. In 2010, we acquired the Illumina Genome Analyzer IIx and successfully performed WGS (bacterial) and RNA transcriptome analysis, with very satisfactory results. Within recent years, we upgraded to a more capable and versatile rand of Illumina platforms, which include the MiSeq®, the HiSeq 4000® and the NextSeq 500®. Of course, in the event of extensive troubleshooting, we maintain a complete technical support plan from Illumina for any problems that may arise.
While GC rich DNA is certainly a problem with Sanger sequencing, it should not be an impediment to NGS Illumina methodologies, which utilizes a different sequencing strategy. However, regions of very low complexity and multiples repeats would still be difficult to correctly sequence.
We provide DNA extraction from the customer’s source materials, library construction and validation, sample run on an Illumina instrument, and data analysis (presented in the Project Report format).
Genomic DNA is the only material suitable for constructing a library for WGS, but we can prepare it from any relevant source material provided by the customer: cells, tissue, spores, blood, etc.
We offer raw Illumina data reads, assembled sequence (contigs and scaffolds), sequence comparison data against one or more reference sequences. We can also customize data analysis to provide whatever information is required by the customer.
We provide total or mRNA extraction from a customer’s source materials, RNA library construction and validation (including qPCR expression level determination for a small set of genes that are the customer’s choice), sample run on the Illumina instrument, and data analysis (presented in the Project Report format).
We prefer to extract RNA in-house, however ACGT will accept prepared RNA samples at the customer’s preference and responsibility. The choice of total or mRNA libraries would be up to the customer.
My organism has significant variability in expression. How can I be certain that we will receive good quality, reliable data?
Variable expression levels is a serious problem, and for this reason that we offer RNA sample validation using RT-qPCR. ACGT can check one or more samples for expression levels of a gene of your choice against some standard housekeeping gene control, and proceed with NGS only if the results are satisfactory. Of course, several transcriptomes can be prepared and tested simultaneously as well.
Yes and no. A basic cDNA pool is generated as an intermediary step in the creation of an Illumina c-Bot cluster library. Some of that pool is used for transcriptome validation, and the rest can be used in subsequent gene expression level confirmation studies using qPCR. However, if the question understands a cDNA library in the context of cDNAs that are subcloned into a plasmid vector and then several thousand or million bacterial colonies are archived, then the answer is no – this would be a separately priced service.
We offer raw Illumina data reads, mapping of reads against ORFs of a reference genome, de novo assembly sequence analysis generating transcript contigs and comparing their sequence against one or more reference sequences to generate sequence homology profiles, relative expression level determination based on mapping frequency of reads per million base pairs, and of course we would customize data analysis to provide whatever information is required by the customer.
In general, the projects for one to a few samples are completed within 2 to 3 weeks following the initiation of library preparation.