FAQs    

• What experience do you and your staff have with next generation sequencing?
  We have extensive experience in preparing various libraries, both RNA and DNA. In 2010, we have obtained the Illumina Genome Analyzer IIx and have successfully performed WGS (bacterial) and RNA transcriptome analysis, with very satisfactory results. Of course, we have complete technical support from Illumina for any problems that may come up.
• Can next generation sequencing be used with GC rich genomes like P. aeruginosa?
  While GC rich DNA is certainly a problem with Sanger sequencing, it should not be an impediment to NGS Illumina methodology, which utilizes a completely different molecular approach.
• What is included in your whole genome sequencing service?
  We provide DNA extraction from customer’s source materials, library construction and validation, sample run on the Illumina instrument, and data analysis (presented in the Project Report format).
• Is purified genomic DNA the only starting material needed for whole genome sequencing?
  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.
• What is the price per bacterial genome and what is included in this price?
  To some degree the price would depend on the specifics of the project, but to give a general idea: DNA extraction and library preparation - $500; sample load per lane of the flowcell -$1500 for a 40x40 paired end read; data analysis - $500 to $1500, depending on the type analysis (re-sequencing vs.de novo) So, for example, if you ran a single bacterial genome for de novo assembly, for a paired end run of 40 cycles each with complete data analysis, the cost would be $3500.
• What options does ACGT, Inc. offer for data analysis for whole genome sequencing?
  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.
• What is included in your transcriptome analysis (RNA-seq)?
  We provide total or mRNA extraction from customer’s source materials, RNA library construction and validation (including qPCR expression level determination for a small set of genes that are customers choice), sample run on the Illumina instrument, and data analysis (presented in the Project Report format).
• Do you require total RNA or mRNA enriched samples?
  We prefer to extract RNA in-house, but will take prepared RNA samples at 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. Can I be certain that we can 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. We 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.
• Is the generation of the cDNA library included in the RNA-seq service?
  Yes and no. A basic cDNA pool is generated as an intermediary step in the creation of 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. But if you mean a cDNA library in a sense that cDNAs are subcloned into a plasmid vector and several thousand or million bacterial colonies are archived, then no, that would be a separately priced service.
• What options do you have available for data analysis for RNA seq?
  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.
• What is the turn-around time for each of these services?
  In general, the projects are completed in between 3 and 5 weeks time after the initiation of library preparation.