A platform to discuss the methods and technologies involved in DNA sequencing.

• Matt Hosek

  Virus Paylod: Suggestions on a vector for larger DNA sequences.

  I'm looking for vectors for DNA sequences larger than 5kB with low association of inflammation/necrosis around injection site and that will deliver and integrate material into (mammalian, en vivo)I'm looking for vectors for DNA sequences larger than 5kB with low association of inflammation/necrosis around injection site and that will deliver and integrate material into (mammalian, en vivo) non-dividing cells. Links to papers or your personal results with any particular virus classes or lines would be greatly appreciated.

  Recent replies ⋅ Show All (2)

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    Matt Hosek replied

    Thank you.There is some concern over the inflammatory response to large infusions of adenovirus and other large, common vectors that are being tested now.

    3 days ago
• Nelson Carvalho Filho

  Who could help me with a protocol to optimize bigdye terminator kit usage? My fragments are no longer than 300bp.

  Sequencing reaction protocol bigdye terminator kit

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    Nelson Carvalho Filho replied

    Thank you very much for indeed for the tips. In fact .5 ul is the limit.

    12 days ago
• Muhammad Ibrahim

  Fusing Contigs?

  Hi, Can any one help me to tell that how I can fuse contigs? I have genome sequence from my lab but three contigs are important for sequence viewer which I want to fuse.

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    Colin F Davenport replied

    There are some excellent discussions and software on seqanswers.com about this topic.

    19 days ago
• Dr. Bharat Bhusan Patnaik

  Novel sequence submission in public databases

  I have sequenced (currently partial nucleotide sequence) and would like to submit the same to the gene and protein databases. I have already posted the N-terminal protein sequence to Uniprot andI have sequenced (currently partial nucleotide sequence) and would like to submit the same to the gene and protein databases. I have already posted the N-terminal protein sequence to Uniprot and received the acession number. Can i update the partial sequence and later full-length sequence with UniProt. Also i want to submit the gene sequence. Please suggest me the process of submission of both.

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    Satyajit Kanungo replied

    Yes sir.

    29 days ago
• Renato Lemgruber

  I`m looking for publications using SAGE for next generation sequencing. Who can help me please?

  I´m working with fish exposed to petroleum using SAGE SOLiD
• Chintsong Lim

  Blast-ed sequencing result from excised DGGE bands

  I'm currently doing soil microbe analysis using DGGE. There are a lot of different species and genus from the blast result. Some of my samples from the blast result give me up to 10 differentI'm currently doing soil microbe analysis using DGGE. There are a lot of different species and genus from the blast result. Some of my samples from the blast result give me up to 10 different species and all with Query coverage and Max ident at 99%. I would like to know how to choose the suitable species represent my sequencing result. Do I have to choose several species or just one?

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    Shreyans Chordia replied

    Your primers have to be more specific to ur 18S target. Check that using primer BLAST. And look at the hits it gives. If in ur hits you get homology for nt's of origin other than soil algae, thenYour primers have to be more specific to ur 18S target. Check that using primer BLAST. And look at the hits it gives. If in ur hits you get homology for nt's of origin other than soil algae, then your primrs are not specific. In that case you should try to design new primers..

    Mar 20, 2012
• Leandro Lemos

  Two adapters keys and pyrosequencing

  Does anyone know why two adapters are often used in pyrosequencing? Thanks!
• Carlos Wolfgang Nossa

  Illumina library prep kits. Nextera vs. TruSeq.

  Has anybody used both Nextera library kits for Illumina sequencing and the TruSeq kits? Was there any difference in the results? We have used TruSeq before, but wanted to switch to Nextera since itHas anybody used both Nextera library kits for Illumina sequencing and the TruSeq kits? Was there any difference in the results? We have used TruSeq before, but wanted to switch to Nextera since it is a more streamlined protocol and there is no mechanical fragmentation step.

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    Carlos Wolfgang Nossa replied

    Here is a good paper that addresses the issue http://www.ncbi.nlm.nih.gov/pubmed/21948828

    Mar 9, 2012
• Javad Jabbari

  How can I identify if two SNPs ( >250KB apart) are located on the same or 2 different alleles, when you don’t have DNA from the family members?

  I am having difficulty to find a method to identify if two or several SNPs are located on the same allele or on two different alleles when they are located far from each other. I don’t have access toI am having difficulty to find a method to identify if two or several SNPs are located on the same allele or on two different alleles when they are located far from each other. I don’t have access to the DNA from the family members. I have searched the literatures but without any luck. In my lab we have only access Applied Biosystems ABI 3730xl 96-capillary DNA analyzer.

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    Javad Jabbari replied

    Thank you very much!

    Mar 3, 2012
• Jack Stockert

  Sample Amplification and GC representation

  I have a quick question regarding the impact of GC representation in my sample preps. I am looking at different sample prep kits to use and one has slightly higher GC representation (upwards of 50%I have a quick question regarding the impact of GC representation in my sample preps. I am looking at different sample prep kits to use and one has slightly higher GC representation (upwards of 50% with 10% variability). Is there a ceiling that I would run into limitations in my results because of this? A colleague has raised significant concern that this is an issue but I do not see an issue as long as the variation is predictable and in a narrow range. Does anyone have any thoughts on this? Thanks a lot.

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    Jack Stockert replied

    yeah, I am trying to push to low level of inputs in the picogram range and the GC issue arose. it is predictable and with little variability from run to run. Siva, you highlight an interestingyeah, I am trying to push to low level of inputs in the picogram range and the GC issue arose. it is predictable and with little variability from run to run. Siva, you highlight an interesting point about the primer-dimer problem, and that is part of what I am seeing. It is limited though and the other results look great. I appreciate the insights.

    Feb 29, 2012
• Aylan Kener Meneghine

  What is better: NCBI (blast n) or Ribosomal Database Project for analysis sequencing 16S rDNA?

  I'm verifying my sequences of cloning in this genbanks, but each give me a result.I heard that RDP is better than NCBI, what can someone say about that ?

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    Ekrem Ayna replied

    There are many junk submissions that had challenged us in NCBI, even submitted under the most reliable institutes. Quickly in and quickly out, this is my advice for NCBI.

    Feb 15, 2012
• Vennobaahshini Venu

  What would be the problem or how to over come it to get 100% match?

  I did PCR amplification for my gene (669bp) and sent it for sequencing. I got my sequencing results and when I blast with NCBI gene bank found that my PCR DNA sequencing only has 99% max identitiesI did PCR amplification for my gene (669bp) and sent it for sequencing. I got my sequencing results and when I blast with NCBI gene bank found that my PCR DNA sequencing only has 99% max identities and 97% query coverage with actual gene. I found that there is few basepair point mutation in comparison to actual gene. Beside that, how to check if its true positive mutate and where the mutation occur eg in the functional protein area.

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    Sa-fa -jnia replied

    Dear Venu, Sequencing of 3 different clones can provide idea rapidly about the source of such mismatches ? If mismatch present in all sequenced clones, then it is not due to Pcr errors!

    Feb 12, 2012
• Kent Kemmish

  Looking for a crowd-sourced estimate: how soon will the first telomere-to-telomere, gapless assembly of a single diploid human genome be produced?

  Crowd-sourcing, completeness of genomes

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    George Church replied

    My guess is 2012. Justifications: 1) Reich et al evidence for an MS gene in the centromere gap of chromosome#1, 2) variations can cause aneuploidy, 3) quality methods can drive cost down. How: MbpMy guess is 2012. Justifications: 1) Reich et al evidence for an MS gene in the centromere gap of chromosome#1, 2) variations can cause aneuploidy, 3) quality methods can drive cost down. How: Mbp stretched DNA +FISSEQ or EM.

    Feb 9, 2012
• Ralph Scheicher

  Epigenetics and DNA sequencing?

  My question to the experts is whether (and if so, how) currently available DNA sequencing techniques can detect epigenetic modifications, in particular methylation. (Any recommendations for furtherMy question to the experts is whether (and if so, how) currently available DNA sequencing techniques can detect epigenetic modifications, in particular methylation. (Any recommendations for further reading on this topic would also be highly appreciated.)

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    Devora cohen-karni replied

    Hi, depending on your purpose and resources, I highly recommend using the MspJI family of modification dependent restriction enzymes for genome wide detection of methylated sites. The enzymes createHi, depending on your purpose and resources, I highly recommend using the MspJI family of modification dependent restriction enzymes for genome wide detection of methylated sites. The enzymes create a double strand cleavage of the DNA 16 bases away from the modified cytosine, forming a 32 base fragment with the fully methylated cytosine in the middle, allowing direct sequencing of the excised fully modified site. The main advantage is that since you only need to sequence the methylated sites you reduce the required sequencing power. The method is described in this paper: http://www.pnas.org/content/108/27/11040.long , and if you are interested in using it, I will be happy to provide additional details.

    Feb 8, 2012
• Alessandra Frau

  Barcoding pyrosequencing

  Hi, I would like to sequence 16s from several environments and barcoding pyrosequencing seems to be a good method to do that saving time and resources. Does anyone have any experience in this? I haveHi, I would like to sequence 16s from several environments and barcoding pyrosequencing seems to be a good method to do that saving time and resources. Does anyone have any experience in this? I have few technical questions and I need someone with a direct experience in issues as sample preparation, quality control etc. Thanks!

  Recent replies ⋅ Show All (6)

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    Cynthe Sims replied

    Here is another method for generating your tagged and linkered amplicons in PCR http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2515157/?tool=pubmed

    Feb 4, 2012