Molecular Minutes

Q&A: NGS Data Analysis 101

Posted by Applied Biological Materials (abm) on November 27, 2019

Here is the full list of answers to questions we've received during the

"NGS Data Analysis 101: RNA-Seq, WGS, and more" webinar:


  1. What file format will I get from sequencing on a MiSeq, FastQ or BCL? Do you provide the BCL files for sequencing?

  2. Do Q30 scores vary on different sequencers?

  3. I don’t have a reference genome but there is a related species that does have a reference genome. Can I use that for alignment/analysis?

  4. For analysis do you use GATK or VarScan2? ​Can I pick which one you use for my analysis?

  5. Do I have to use JBrowse? ​Does anyone still use Gbrowse?

  6. What happens if I don’t normalize my RNA-Seq data using FPKM?

  7. What should I do if I do not have appropriate “control” samples for RNA-Seq? 

  8. Can StringTie be used to identify fusions, or do I need to use another program for this?

  9. If I want to identify alternative-splicing transcripts from RNA-Seq data, do I have to do anything differently for sequencing?

  10. If I did single-end RNA-Seq, do I still normalize the data the same way?

  11. Can you do custom analysis? I want standard and custom analysis for a project.

  12. For RNA-Seq data alignment, can I use BWA-MEM as the aligner or can I use something else, like STAR (Spliced Transcripts Alignment to a Reference) software?

  13. Do I need to use replicates for my RNA-Seq experiment?

  14. Do I have to normalize my data before using DESeq2 for looking at differential gene expression? Or can I input FastQ data and the software can process it anyway?

1. What file format will I get from sequencing on a MiSeq, FastQ or BCL? Do you provide the BCL files for sequencing?

Our standard deliverable for all sequencing are FastQ files for all projects. If you would prefer to receive BCL files, please let our team know before order placement to discuss further! In most cases, we can provide these, depending on your project, and the sequencing platform.

 

2. Do Q30 scores vary on different sequencers?

Yes, the Q30 scores generally differ. For instance, NovaSeq > HiSeq > NextSeq > MiSeq, although the choice of sequencer will affect Q30 scores less than other factors such as read length, sample quality, or even the actual sequence for the sample.


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Topics: Next Generation Sequencing, NGS, Data Analysis

Q&A: Getting Started With Whole Genome Sequencing

Posted by Applied Biological Materials (abm) on November 7, 2019

Here is the full list of answers to questions we've received during the "Getting Started with Whole Genome Sequencing" webinar: 

    1. How many cells should I use for DNA Isolation for whole genome sequencing?
    2. There is not a reference genome for the species I study, but there is a sequenced genome for a related species... Can I use that?
    3. How much coverage do I need? How do I figure this out? Help!!
    4. For Plasmid Verification, do I have to provide a reference sequence or can you do de novo assembly?
    5. For mtDNA-Seq, do you have to start with isolated mtDNA? Can I just submit purified gDNA?
    6. How many samples should I submit for WGS?
    7. If my sample has RNA contamination, can you do anything to remove it?

 

1. How many cells should I use for DNA Isolation for whole genome sequencing?

For most projects, we start with ~1 million cells. This ensures we can extract enough DNA to perform full QC, library preparation, and have enough material left over in case any step needs to be repeated.

 

2. There is not a reference genome for the species I study, but there is a sequenced genome for a related species... Can I use that?

Even for very closely related species, there can be many differences between genomes. A related species can possibly be used as a guideline for assembly, but it cannot be used as a reference. 

An analogy that may better help you to understand this is a jigsaw puzzle. Think of two different puzzles, where both depict similar, but different castles. For one puzzle, you know what the completed image should look like, but for the other, you do not. If you use the reference puzzle as a guideline, you may be able to approximate the puzzle where you do not know what the completed image looks like, but you don't know how accurate this may be.  


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Topics: Next Generation Sequencing, NGS, Whole Genome Sequencing, WGS

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: The Beginner's Guide to RNA-Seq Webinar

Posted by Applied Biological Materials (abm) on August 7, 2019

Here is the full list of answers to questions we've received during The Beginner's Guide to RNA-Seq webinar:

    1. Can you explain in a bit more detail why sequencing adaptors are used?
    2. Once the sequencing is done, how do I trim the adaptor sequence from the sequencing result? That is, how do I make sure the sequence information I have is for my target sequence, but does not include extra information, such as from the adaptors?
    3. What is the difference between gene profiling and gene expression?
    4. If I’ve done my experiment and have the raw sequencing data, but I do not know how to perform the analyses that I would like, can I give you the data and have you do the analysis?
    5. Do you know where I can get RNA seq data for cancer genomics research?
    6. Is it possible to sequence both circRNA and mRNA in the same RNA-Seq project? Is there an enrichment step or is this done through bioinformatics?
    7. How long does it take to do library prep, quality control, and sequencing?
    8. Can I submit one sample and use it for both RNA-Seq and miRNA-Seq? Or do I need to submit separate samples for those?
    9. Can I get one-to-one expert help if I want to have a deeper knowledge in all steps that are involved in sequencing?
    10. What happens if my samples fail QC?
    11. Can you explain a bit why clustering is necessary in the sequencing process?
    12. Do you have any representatives or distributors in Brazil or in the Czech Republic?
    13. How do I go about generating a heat map and principal component plot for my data? Is this something that you can help me with?

1. Can you explain in a bit more detail why sequencing adaptors are used?

The adaptor is important for the DNA fragment to bind to the sequencer; without this step, the sample would be washed away before the sequencing reaction begins.

The adaptor also includes a sequence which is important for the sequencing primer to anneal. Without this annealing, the sequencing-by-synthesis reaction could not occur.

Finally, when you sequence more than one sample at a time (called multiplexing), you need unique sequences in each adaptor to be able to distinguish Sample 1 from Sample 2 from Sample 3, etc. The adaptor sequence also contains this unique, sample-specific barcode, that allows you to tell which sequencing reads belong to which sample.


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Topics: Next Generation Sequencing, NGS, RNA-Seq

Next Generation Sequencing (NGS): What are Sequencing Coverage and Mapping Coverage?

Posted by Applied Biological Materials (abm) on June 13, 2019

If you're starting a sequencing project, you may encounter terms such as "Sequencing Coverage" or "Coverage Mapping". In this blog post, we'll explain how you can estimate these numbers for your own project!

 

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Topics: Next Generation Sequencing

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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