Viral Genome Sequencing_Library Preparation

[mukeshwar]

Hello,

I have started working on viral genome sequencing on illumina MiSeq platform. For RNA viral genome i followed the truseq RNA sample preparation protocol for library preparation after dsCDNA conversion of total viral RNA but i am little confused about the sample preparation of those viral sample that contain DNA + RNA (total nucleic acid). Can anyone suggest what should be the work-flow and protocol for library preparation???

[bbeitzel]

Do you want to sequence RNA virus genomes out of your total nucleic acid mix? If so, you can simply DNase you total nucleic acid prep and then proceed with the RNA sample prep you are already doing.

[ismail selim]

ı started working on H1N1 viral genom sequencing, to get information noncoding regions (A new approach to determining whole viral genomic sequences including termini using a single deep sequencing run) ı try this protocol but its to long and its not working. Can anyone suggest what shouldnt be working??

[adam.geber]

Can you describe your current protocol with more detail? Unless you're starting from influenza culture samples you won't get good coverage of your genome without further enrichment for viral nucleic acids. I can point you towards resources for influenza deep sequencing once I know what you're currently working with.

[ismail selim]

I starting with 119 ng/µl broad rage RNA kit, use for ribosomal RNA removing NEbnext depletion kit, dynabead for mRNA removing, concentrate with using RNA clean &Concentrator Zymo. Than fragmentation with Mg++2 (Nebnext fragmentation kit) for more efficient effecitive data representing 3' and 5' ends. The sample were incubated at 84◦C, 7 min. Following fragmentation the terminal ends of the RNA wereprepared for adaptor ligation. Antarctic Phosphatase (New EnglandBiolabs, Ipswich, MA, USA).Next, samples were treated with Polynu-cleotide Kinase (New England Biolabs, Ipswich, MA, USA) to add a 5phosphate, according to the manufacturer’s protocol. FragmentedRNA was cleaned up again with the RNeasy MinElute Spin ColumnKit (Qiagen, Germantown, MD, USA) according to the manufac-turer’s protocol.Adapter ligation was performed in two steps usingreagents from Illumina’s TruSeq Small RNA Sample Preparation kit(Illumina, San Diego, CA, USA). First, the RNA 3Adapter T4 RNA ligase 2, truncated (New England Biolabs, Ipswich,MA, USA.Next, the 5adapter was ligated to theRNA T4 RNA ligase wereadded to the RNA 5adapter solution.PCR enrichment, usingIllumina’s TruSeq Small RNA Sample Prep Kit protocol and reagents(Illumina, San Diego, CA, USA). Lastly, the samples underwent twofinal cleanups with Agencourt AMpure XP Beads (Beckman Coul-ter, Irving, TX, USA) according to the Illumina’s sample preparationprotocol and too much different kit used and ı dont find what is wrong with. '' A new approach to determining whole viral genomic sequencesincluding termini using a single deep sequencing run''