Originally posted by Genohub
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70 16S V4 library preps and MiSeq sequencing services can be ordered directly by clicking on "start project". Submitting your project allows you to communicate shipping and sample logistics with the provider directly using our project dashboard interface. Let me know if you have any questions.
- Genohub
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Originally posted by Genohub View Post70 16S V4 library preps and MiSeq sequencing services can be ordered directly by clicking on "start project". Submitting your project allows you to communicate shipping and sample logistics with the provider directly using our project dashboard interface. Let me know if you have any questions.
- Genohub
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Hi Nirajlincoln,
In most microbial species the 16S fourth hypervariable (V4) region is approximately 254 bp. 254 bp is a read length that can easily be read using paired-end Illumina sequencing, making it a popular metagenomic target. You can read through the entire V4 region using a HiSeq 2x150 Rapid Run run or MiSeq runs of at least 2x150 (2x250 and 2x300 is also possible, but will give you overlap in each read). For 70 multiplexed samples you will get between 80,000-400,000 reads per sample (depending on the MiSeq version) which is more than enough for classification and profiling. Of course, if you are interested in looking at extremely rare taxa from similar environments, deeper sequencing will help. Contact us using our consultation form and we'll be happy to refer you to 16S V4 library and sequencing services. We're also happy to discuss your analysis requirements.
- Genohub
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Hi!
I noticed that several people have asked about a facility that does the library/PCR prep, etc. for the MiSeq and 16S. We do that here at our facility, Selah Genomics. We are located at the University of South Carolina and I am the bench tech that handles all of the library prep, PCR/Amplicon prep, etc. for all of our platforms that we currently have. We have the MiSeq. I have extensive experience in preparing 16S samples using the Caporaso method along with all of the current Illumina kits offered on the MiSeq. Please feel free to contact me at [email protected] if you are in need of a quote for services and I will be glad to get that started for you.
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long miseq technique
Hi, a while back I read an article that accomplished the 2x300 time by taking adding extra reagents from one 2x250 kit to another 2x250 kit. I need to find that reference but I have absolutely nothing else to go off of to search. Anyone out there know what paper I'm talking about? Thanks!
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Hi,
What are the sequencing primers used to sequence the V4 hypervariable region of 16S rRNA gene? For amplification, I have used 515f/806R.
I am getting my samples sequenced by sanger sequencing. But when people at sequencing facility try to sequence the same sample using Illumina Miseq, it is a failure.
Please help
Thanks,
Richa
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Hi Richa,
The sequencing read primers will depend on how you designed your upstream and downstream PCR primers. Are you using the primers here: http://www.earthmicrobiome.org/emp-s...protocols/16s/ ? Remember that you can't rely on the Illumina seq primers if your PCR primers are custom. Were your customer seq primers added appropriately to the right MiSeq reservoirs ?
Other considerations: check the % phiX spike and change your index cycles so you're reading your entire barcode sequence.
- Genohub
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Hi Richa,
Your V4 primers not only flank the V4 domain but also contain Illumina’s flow cell sequence. You need the custom sequencing primer to properly read your final amplicon. There are three seq primers: read 1, read 2 and the index read. The index primer is designed to only read the barcode, and is specific to Illumina workflows.
PhiX is a adapter ligated control library that comes from the PhiX genome. In this application it's used to increase your sample diversity or balance your CG / AT biased amplicon samples. Since you're dealing with amplicons, the start position of each read is going to be similar, which can be a problem with Illumina seq reads. In earlier MiSeq software users had to spike in as much as 30-40%, now with the latest Illumina software updates you can get away with a 5-10% spikein.
Send us an email at [email protected] if you'd like us to recommend a sequencing provider experienced in 16S V4.
- Genohub
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FYI. The latest RTA software is much better than a year ago. I am currently running 3 amplicons with 10% phiX at 700k/mm^2. It's a nano, but Im getting 1M reads with 95% passing filter and 99% Q30. I can honestly read the primer sequences from the intensity chart. I do not think it is necessary to use a custom seq primer if you have several amplicons in the mix.
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Hi Richa
We have quit using the V4 domain, as our internal testing has been less the encouraging regarding its ability to accurately recover the identities of known human pathogens in a mock community. Instead we are using V1-V3 (or V1-V2) on MiSeq (we also offer V3-V5 and V3-V4) which much more closely recover the information we expect from our Mock communities. We offer full service sequencing from DNA-analysis on MiSeq, so feel free to contact me if you need any help or pricing - [email protected]
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Originally posted by csquared View PostIt provides a "pad" for the primers to anneal for the sequencing versus amplification primers. Hard to explain in perfect detail in text but if you diagram the amplicon and draw where each primer starts and ends, the role of the pad is a bit easier to see. The primer pad is the same as the 5'-end of the sequencing primers (read 1 and read 2) and the same as the 3' end of the index read primer.
Note that the 2011 Caporaso PNAS paper may have a typo in the sequences shown in Figure 1. Unless I'm missing something, the Read 2 sequencing primer and the Index read primer are not compatible with each other. One of them has a typo and I think the Index sequence is correct and matches the primer sequence list provided in supplemental material in that paper or one of his other recent papers.
Is Caporaso's P5 and P7 sequences compatible on Miseq flowcell too?
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MiSeq for 16s sequencing of V4
Hello! I will like to do metagenomic sequencing of the V4 region. I will like to use 2 x 250 cartridges, Nextera XT kits and Illumina manufactured indexes. Is there a detailed protocol for doing this? Illumina has a related Application Note, but it provides few details. Thanks
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