Mon, 12/30/2013
Customer indicates that he wants to conduct a RNA-seq study using Illumina’s Miseq sequencer.
Mon, 12/30/2013 at 9:32 AM
AccuraScience LB: Do I understand it correctly that you are working on a species with existing reference genome? If you are unsure, please tell me the species name, and I will find this out for you. This is an important question because the RNA-seq analysis methods for a species without a reference genome are very different from the ones with a known reference geome. The cost of the former analysis is one order of magnitude higher than that for the latter.
You indicated Illumina's MiSeq sequencer in the last inquiry, which is puzzling because people typically do not use MiSeq for RNA-seq studies. Could you confirm that this is in fact what you plan to do? And, could you tell me the sample numbers you are planning for the RNA-seq experiments?
Mon, 12/30/2013 at 9:48 PM
Customer: The genome sequences is available in public domain. As I am new to RNA-seq field, and Illumina Miseq was suggested by someone. However, may I ask you to suggest the proper sequencer/methods to use? We have 6 samples (all bacterial).
Tue, 12/31/2013 at 2:51 PM
AccuraScience LB: Illumina's Miseq sequencers were designed for lower-throughput experiments, e.g., bacteria genome sequencing. The disadvantages of MiSeq, as compared to HiSeq, include (1) its per-base cost is substantially higher than that for HiSeq (could be ~10 times higher), and (2) they are not as popular as HiSeq, which means it is not as easy to find a sequencing facility equipped with MiSeq than it is for HiSeq, making shopping around more difficult. In contrast, practically all sequencing facilities today are equipped with HiSeq sequencers.
However, given that you have only 6 bacterial samples, unless you can pad in samples from other projects, the saving you get using HiSeq will not be very significant. Here's why: For bacterial RNA-seq experiments, the recommended sequencing coverage is 5-10 million reads per sample (http://www.ncbi.nlm.nih.gov/pubmed/23270466), thus 15-30 samples can be fit into one HiSeq lane. If you have other samples for other projects besides these 6 samples, you could use one lane to sequence them all (up to 5 projects worth). Otherwise you will have to use the whole lane for your 6 samples. This sequencing experiment will cost you about $3000-4000 plus barcoding cost (could be another $2000 depending on the number of samples you would want to pool together).
If you would go the MiSeq option (given that you can find a MiSeq sequencer), each MiSeq run can produce 50 million reads, so your 6 samples could fit in one MiSeq run snugly. One run of MiSeq will cost about $2500-3500, plus the barcoding cost.
Please check this article http://www.ncbi.nlm.nih.gov/pubmed/21863565 for sample preparation procedures.
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Note: LB stands for Lead Bioinformatician. An AccuraScience LB is a senior bioinformatics expert and leader of an AccuraScience data analysis team.
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