Molecular Minutes

Q&A: Using NGS for CRISPR Validation, Antibody Screening, & Metagenomics

Posted by Applied Biological Materials (abm) on October 25, 2019

Here is the full list of answers to questions we've received during "Using NGS for CRISPR Validation, Antibody Screening, & Metagenomics" Amplicon-Seq webinar:

    1. If I have already done sequencing, but need help with the analysis, can you help me?
    2. What is the largest size of amplicon that I can have sequenced?
    3. What is the typical turnaround time for Amplicon-Seq, and can you offer an expedited service if I am in a rush?
    4. Can I do Amplicon-Seq to look for off-target effects as part of CRISPR validation?
    5. If I’m interested in metagenomics sequencing, can I do Amplicon-Seq for targets other than 16S, like fungi or other eukaryotes?
    6. Do you guarantee a minimum number of reads for projects or samples?
    7. When discussing coverage, does 50X mean 50 reads?
    8. I'm interested in CRISPR validation and metabolomics. Can I use Amplicon-Seq to look at metabolites?
    9. Is Krona the software used for identify species for metagenomic sequencing? If not, how do we determine the specific microorganisms present in our samples?
    10. Can you help design gRNA's for my CRISPR experiment?
    11. What are some considerations for using the HiSeq 4000 platform?
    12. When discussing read length, what does 2x300 bp PE mean?
    13. If I have already done sequencing, but need help with the analysis, can you help me?

 

 

1. If I have already done sequencing, but need help with the analysis, can you help me?

Our in-house bioinformatics team can assist with nearly any type of analysis (in as little as 1 week!) based on your project needs. In the near future, we will be launching a dedicated Bioinformatics Service for researchers, but if you have a project where you need help with analysis, you can contact our NGS team at ngs@abmgood.com. One of our team members can then help you with analysis you are interested in completing!


 

2. What is the largest size of amplicon that I can have sequenced?

For most projects, we can sequence amplicons that are <600bp. If you have an amplicon that is larger than this, please let us know, and we may be able to work with you on a customized solution for your project.


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Topics: CRISPR, Next Generation Sequencing, NGS, Amplicon-Seq, Metagenomics

Q&A: Essential Guide to Becoming a CRISPR Cas9 Expert Webinar

Posted by Applied Biological Materials (abm) on May 30, 2019
Here is the full list of answers to questions we've received during the abm CRISPR webinar:

  1. What about experimenting with HeLa Cells? How do you go about performing a CRISPR knockout sequence on HeLa cells?
  2. You mention designing sgRNAs, what kind of controls can we use for our knockout experiments?
  3. Are there ongoing efforts to reduce the cost of next gen sequencing?
  4. Would a custom sgRNA be needed to use CRISPR/Cas9 in zebrafish?
  5. How expensive would it be to custom design sgRNA?
  6. What are the differences between genome editing in primary vs. stem cells?
  7. While using an all-in-one Cas9 and sgRNA vector, should the selection markers for the colony be two different markers or do we only need to use a bacterial selection marker?
  8. How is CRISPR better than siRNA? I have been using siRNA for my past studies.
  9. Will saCas9 become more popular as viral vectors become more widely used?
  10. What abm products or services are available for species other than mice and human, for example, for chicken?
  11. I've read that there are shortages for viral vectors right now. Is this an issue for you? Does abm generate viral vectors in-house?
  12. What about inducible Cas9 models? How do they compare to constitutively expressing Cas9 models?
  13. What about plant systems? abm has sgRNA libraries for mammals but not for plants, although it is in high demand.
  14. Can we depend on the MIT specificity score while selecting sgRNA to minimize the off-target effects?
  15. Can CRISPR be used on long noncoding RNA?
  16. Does abm perform cell line knockouts for plants as well?
  17. What is the common method to extract Cas9 from a bacterium? Using transfer vector and affinity chromatography?
  18. Can I use CRISPR to knock down virulence genes of pathogenic bacteria - for example, Shigella flexneri
  19. Are there specific recommendations to consider when using CRISPR with microRNA?

1. What about experimenting with HeLa Cells? How do you go about performing a CRISPR knockout sequence on HeLa cells?

There are no problems doing CRISPR in HeLa cells, as long as your target sequence is found in the genomic DNA sequence, and the delivery method you use has high efficiency in your cell line. In our experience, lentivirus works very well with HeLa cells. In fact, abm has Cas9 Expressing HeLa Cells (Cat. No. T3254) to help you get started.


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Topics: CRISPR

Molecular Minutes

Educational resources for life scientists and interviews with scientists/science communicators in the field.

For more in-depth articles, check out our knowledge base, which covers topics such as CRISPR, Next Generation Sequencing, PCR, Cell Culture, and more.

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