4) Next Generation Sequencing (NGS) - Data Analysis
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- Опубліковано 21 лип 2024
- What is covered in this video:
➜ Previous videos in our Next Generation Sequencing (NGS) series describe the theory and technology of NGS platforms ( • 1) Next Generation Seq... ), and the steps of library preparation for sequencing on the Illumina platform ( • 2) Next Generation Seq... ). In this installment we describe some of the common formats of NGS raw data and software that can be used for downstream analysis.
For more information on Next Generation Sequencing analyses and for a list of the sources used, please visit:
➜ Knowledge Base: info.abmgood.com/next-generat...
You can also watch our free webinar on NGS Data Analysis here:
➜ • NGS Data Analysis 101:...
Watch the other videos in this series on NGS:
➜ Introduction: • 1) Next Generation Seq...
➜ Sample Preparation: • 2) Next Generation Seq...
➜ Coverage & Sample Quality Control: • 3) Next Generation Seq...
➜ NGS Playlist: • 1) Next Generation Seq...
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Even though this video was 8 years ago, it still seems great. I love it. Thanks a lot
1. Raw Data Output (.bcl ➔ fastq) 0:37
2. Sequence Alignment 2:20
1) BWA
2) Bowtie2
3) Maq
4) Stampy
5) Novoalign
3. Variant Calling 4:28
4. Additional Software & Tools 6:12
1) FastQC
2) Picard
Very good, clear explanation. Wish I'd found this sooner rather than scratching my head about some of the things explained here! Great work.
Thank you so much! Glad to hear that you found the video helpful.
THANK YOU , good video for beginners in NGS
Awesome, that's great to hear!
Thank you. I had a trouble reading methodology for research papers. Now i get it .
Hello Danny,
Thanks so much for watching! We are glad to hear that our video was helpful. Please stay tuned for upcoming videos as well!
very simple...easy to understand thankyou
Thanks for your comment! Please let us know if you have any questions, we'd be happy to help. You can also check out our knowledge base for more info: goo.gl/Ce0M4O.
Fantastic video, thank you
You're welcome! :)
awesome video!! very useful for NGS starter
Hi Chengyu, thanks for your comment - glad you enjoyed it!
what does phasing weight and prephasing weight denotes and also kindly explain what is slope/offset ratio for phasing and prephasing for a run in HiseqX instrument
Hello Harisankar,
This is a difficult one so we've contacted Illumina to see if they can explain. We are waiting for their reply and will let you know once they provide an explanation.
Awesome
nice
Dear abm, thank you for these informative videos. Can you explain to me what does the Cluster Density between 200
, Clusters Passing Filter and estimated Yield for the run mean?
You're welcome, and thanks for checking out our videos! To answer your questions:
1) The Illumina sequencing workflow is based on 3 simple steps: libraries are prepared from virtually any nucleic acid sample, amplified to produce clonal clusters, and sequenced using massively parallel synthesis. The density of clonal clusters has a large impact on sequencing performance in terms of data quality and total data output.
Cluster density is a critically important metric that influences run quality, reads passing filter, Q30 scores, and total data output. While underclustering maintains high data quality, it results in lower data output. Alternatively, overclustering can lead to poor run performance, lower Q30 scores, the possible introduction of sequencing artifacts, and-counterintuitively-lower total data output. Performing a run at optimal cluster density involves finding a balance between under- and overclustering. The goal is to sequence at high enough densities to maximize total data output, while maintaining low enough densities to avoid the negative effects of overclustering.
2) The percentage of clusters passing filter (%PF) is an indication of signal purity from each cluster. Overclustered flow cells typically have higher numbers of overlapping clusters. This leads to
poor template generation, which then causes a decrease in the %PF metric. Reduced yield (gigabases [Gb] per flow cell) is a byproduct of lower %PF.
For these 2 topics, you can learn more from this helpful document from Illumina: www.illumina.com/content/dam/illumina-marketing/documents/products/other/miseq-overclustering-primer-770-2014-038.pdf
3) "Estimated yield for the run" is the estimated sequencing output for a given sequencing run.
@@abmgood thank you very much. I really appreciate your assistance
@@rnh1297 You're welcome! :)
Is there any english subtitle for this video?
Hi Moslem, Thanks for leaving a comment! Yes, English subtitles are available. Simply click on the gear icon on the bottom right of the video. You should see an option for "Subtitles" under which there should be an English option!
I did bowtie2 and I got Sam files what is the next step?
When do we have vcf file?
Sorry, I don't quite understand-could you clarify your question?
Applied Biological Materials - abm Here is the situation，I ask a sequencing companay for raw data，they said because of business secret, they can only release vcf file of pannel sequencing to me. So, what is vcf file and when do we get the vcf？ After fastq?
Hi @@freeloyaltyscenery237, the VCF file is generated after alignment of your FASTQ data to a reference genome and running bioinformatics to determine any differences/variants/mutations in your sample's FASTQ data compared to the reference genome sequence.
Here is a good resource that explains more about what a VCF file is, how it is generated, and how to interpret the information on it:
gatkforums.broadinstitute.org/gatk/discussion/1268/what-is-a-vcf-and-how-should-i-interpret-it
scRNA seq is treading. It will be better if you good upload one.
Hi Jayalal, thanks for the suggestion! We'll add it to our list of possible future videos.
thank you for a very nice and helpful video, I want some more help from you.
Pls provide your email id, so, I can put my queries regarding my research area of
Hello there,
Please contact us at technical@abmgood.com! Thank you.