SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa



Similar Threads
Thread Thread Starter Forum Replies Last Post
A small confusion about Illumina Amplicon Sequencing Protocol discoveradnan Illumina/Solexa 2 11-25-2015 11:36 PM
Amplicon sequencing of a really long single size 2.2 kb amplicon library TompaB Illumina/Solexa 0 02-22-2015 12:39 PM
Fungal ITS amplicon sequencing (NexteraXT vs TruSeq Custom Amplicon) chapadosj Metagenomics 0 06-07-2013 07:59 AM
FLX vs Titanium, Lib-L vs Lib-A, Amplicon Libraries = confusion SeqTruth 454 Pyrosequencing 4 03-03-2012 12:08 PM
Titanium amplicon fusion primer confusion Phimes Sample Prep / Library Generation 3 01-17-2011 06:53 AM

Reply
 
Thread Tools
Old 02-23-2016, 04:21 AM   #1
moistplus
Member
 
Location: Germany

Join Date: Feb 2016
Posts: 40
Lightbulb Confusion about amplicon sequencing

Hi,

Iam a bit confused about how amplicon sequencing work.

Based on this figure:

http://www.illumina.com/content/dam/...n_workflow.jpg

1) I don't understand what is the custom probe is ? Is it the primer ?

2) Then, what are you refer as an adapter ? Is it the Index or 'P7' or Index+P7 ?

3) At the end, with a MiSeq for example, what is the composition of the reads ?
Primer+interest sequence ? or the adaptor is include too ?

Thanks for your answers
moistplus is offline   Reply With Quote
Old 02-23-2016, 05:27 AM   #2
microgirl123
Senior Member
 
Location: New England

Join Date: Jun 2012
Posts: 199
Default

This confuses a lot of our investigators too! TruSeq custom amplicon is a way to sequence many different regions of interest at once (I think it's aimed at clinical studies on things like cancer) as opposed to sequencing one or two different amplicons (such as 16s or 18s which are commonly used for metagenomic studies). If you're only interested in sequencing one or two different amplicons then you need to look into Illumina's 16s metagenomic library preparation protocol, which can be adapted for use with any amplicon.

https://support.illumina.com/content...15044223-b.pdf

Which are you interested in?
microgirl123 is offline   Reply With Quote
Old 02-23-2016, 09:10 AM   #3
moistplus
Member
 
Location: Germany

Join Date: Feb 2016
Posts: 40
Default

I'm interesed in sequencing just a region of dna (not metagenomics studies).
moistplus is offline   Reply With Quote
Old 02-24-2016, 12:48 PM   #4
niroshan
Member
 
Location: Germany

Join Date: Jun 2011
Posts: 10
Default

1) Yes, these are you region specific primers, adjacent to your region of interest

2) Adaptors are the portion of your library read fragment that will attach to the flow cell surface. The flow cell surface has the complementary sequence of these adaptors (called P7 and P5).
Adjacent to the adaptors are the index sequences, so in case of a single-indexed library you will have P7+Index.

3) For single-index: P7 + Index + Forward Primer + Region of interest + Reverse primer + P5

For Dual-index: P7 + Index 1 + Forward Primer + Region of interest + Reverse primer + Index 2 + P5
(which is depicted in the image you linked above)
Attached Images
File Type: png read.png (104.2 KB, 31 views)
niroshan is offline   Reply With Quote
Old 02-24-2016, 01:33 PM   #5
moistplus
Member
 
Location: Germany

Join Date: Feb 2016
Posts: 40
Default

Thanks! It's clear now.
moistplus is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 11:07 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO