SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
Exact duplicate reads/readnames/quality/tiles in Novaseq FASTQs ECO Illumina/Solexa 3 02-28-2019 02:25 PM
very low coverage WGS on Novaseq 6000? msincan Illumina/Solexa 3 11-30-2018 03:30 AM
pooling reads for error correction ilya Bioinformatics 0 04-25-2018 09:33 AM
Pooling reads from different libraries jg3197 RNA Sequencing 3 05-18-2015 08:53 AM
Diff expression: pooling reads of replicates/treatments for de-novo assembly CarlosVM RNA Sequencing 2 01-10-2014 03:05 AM

Reply
 
Thread Tools
Old 05-06-2019, 05:35 PM   #1
KB*
Member
 
Location: UK

Join Date: May 2018
Posts: 15
Default Pooling libs. for Novaseq. How many reads/coverage?

Hi,

I need to pool 10 multiplexed libraries with different insert sizes (from 245 to 285 bp). Two of the libraries are inputs. The rest are IPs. We ordered 50 million reads for each library, 150 paired ends reads.

The core now is going to mix the libraries. The core asked about the concentration I want my libraries to have at. I first planned to have the libs at equal molar concentration, but now I doubt. We sequence samples of the whole human genome (inputs) and fractions of that genome (IPs). It looks like I have to have higher coverage for the whole genome (inputs). That human genome has multiple genetic aberrations.


Different resources recommend different coverage for ChIP:
100x [1] or 10-40X [2]. The recommended number of reads is 20-40 millions [3].

If I calculate coverage,
C=L_reads*#reads/HGenome_length, where
L_reads equal to 150*2=300bp;
# reads, number of reads, 50*10^6;
HGenome_length - length of human genome is ~3*10^9 bp;
C = (300*50 10^6)/(3 10^9) = 5
Assuming I have only 50% of useful reads, it is just 2.5X...

It feels wrong as number of reads is huge.

I can remove 5 of the samples. In theory, it increases the coverage two times.

I can increase input concentration. How how much?

I am confused, please help.

[1] https://www.illumina.com/content/dam...alculation.pdf
[2] https://genohub.com/next-generation-...-guide/#depth2
[3] https://genohub.com/recommended-sequ...y-application/
KB* is offline   Reply With Quote
Reply

Tags
coverage, pooling libraries, reads

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 11:24 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO