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

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