Molecular Minutes

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

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

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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.


 

3. What is the typical turnaround time for Amplicon-Seq, and can you offer an expedited service if I am in a rush?

For most Amplicon-Seq projects, we can complete these in approximately 2-4 weeks. We may also be able to offer an expedited service of 1-3 weeks, depending on upcoming sequencing runs. It is always best to contact us in advance to coordinate a project where you are in a rush.


 

4. Can I do Amplicon-Seq to look for off-target effects as part of CRISPR validation?

Amplicon-Seq can be used to look for edits in your gene of interest, based on your sgRNA’s targets, but to look for off-target effects, you will need to sequence the whole genome and compare an edited sample to a control. Luckily, we offer Off-Target Analysis by Whole Genome Sequencing, as well, and we still have a promotion on discounted analysis for WGS projects. To take advantage of this, contact our NGS team at ngs@abmgood.com to start!


 

5. If I’m interested in metagenomics sequencing, can I do Amplicon-Seq for targets other than 16S, like fungi or other eukaryotes?

Yes, you can essentially use Amplicon-Seq for any target. For metagenomics studies in fungi, for example, we would generate amplicons that target the ITS regions, or 18S for eukaryotic samples.


 

6. Do you guarantee a minimum number of reads for projects or samples?

For NGS services, there is often sample-to-sample variation. To help with this, we often specify a target number of reads per sample, on average, as well as a guaranteed minimum number of reads. We can also adjust your sequencing project depending on your own unique research needs.


 

7. When discussing coverage, does 50X mean 50 reads?

With sequencing, we often discuss reads, coverage, AND read pairs. For example, 1X coverage would represent 1 paired read (1 forward read and 1 reverse read). 50X coverage therefore would represent 50 paired reads, (50 forward and 50 reverse reads).


 

8. I'm interested in CRISPR validation and metabolomics. Can I use Amplicon-Seq to look at metabolites?

Amplicon-Seq can be used to determine the types of edits that occur in your target gene after a CRISPR experiment, and Whole Genome Sequencing can be used to look at Off-Target Effects elsewhere in the genome. Neither option can assess metabolites as the starting material, though, as all NGS requires DNA or RNA as the starting material, not 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?

Krona is a way to visualize data results, rather than software for species recognition. There are many different databases and software, though, that can assist with this, depending on your project needs.


 

10. Can you help design gRNA's for my CRISPR experiment?

Of course! We offer custom sgRNA design services with our CRISPR vectors and viruses. Simply pick your preferred system, provide the gene ID for your gene of interest, and our technical support team can assist you. Learn more at https://www.abmgood.com/Custom-CRISPR-sgRNA-Lentiviral-Vector-Virus.html and email our team at technical@abmgood.com once you are ready to proceed!


 

11. What are some considerations for using the HiSeq 4000 platform?

The HiSeq platform offers one of the most high-throughput sequencing options available on the Illumina platform, with the highest levels of accuracy. As the platform can generate massive amounts of data, many researchers do not have enough samples to efficiently sequence on a HiSeq run, and those that do sequence on this platform may feel overwhelmed by the volume of data generated. Each platform has a number of important considerations that a researcher should review prior to beginning a new project, but careful planning and project design can take care of most concerns.


 

12. When discussing read length, what does 2x300 bp PE mean?

This means that the read length would be 300nt in each direction (1 forward read, of 300nt in length, and 1 reverse read, also 300nt in length). Together, this is considered a read pair of 2x300 nt/bp.


 

13. 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!


 

Got more questions? Feel free to contact our Technical Support Team at ngs@abmgood.com or leave your question in the section below. We'll be happy to help.

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

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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