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Old 10-05-2020, 02:43 PM   #1
rozita
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Location: UK

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Exclamation ChIP-Seq Library Prep - help

Hi everyone,

I'm supposed to do a lot of ChIP-Seq experiments in the coming weeks, I've optimized my first set in terms of qPCR, so my ChIP should be working. I'll also be doing the library prep using the NEBNext Multiplex Oligos for Illumina (Set 1, NEB #E7335) along with the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645L). The samples will be sequenced on Novaseq 6000 S4, PE150, 30 million reads / sample.
I have the following conditions:
Untreated - IP, IgG, INPUT
Treated - IP, IgG, INPUT

I've got a couple of questions now regarding the library prep:
1. Is there a maximum number of libraries that I can pool together? In other words, are there any restrictions or things that I need to consider before pooling them?
What I know so far is that they need to have unique indices and since I'm using NEBNext Mulitplex Oligos for Illumina Set1, there are 12 different indices so I was considering having every 6 libraries pooled together (1 biological repeat).

2. Is it better to have the 2 biological replicates' libraries pooled together or to have each replicate's set separately?

3. Do I need to use the same quantity of DNA for IP, IgG and INPUT when I start the library preparation step? I will quantify them by Qubit before that. I usually get around 1-1.5 ng of DNA for the IP & IgG samples and approx. 100-200 ng DNA for the input. If I use the same quantity of DNA, would I still sequence the input for the same depth as the IgG & IP samples?

Thank you in advance!
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Old 10-05-2020, 03:10 PM   #2
nucacidhunter
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There is no limit on number of libraries (up to 384 is supported by Illumina) for pooling except that they should have unique index for post-sequencing demultiplexing.

One lane of S4 flow cell outputs 2-3B reads so you can pool at least 66 libraries for sequencing (30M each). It is better to use NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer set1 or set2) for correct demultiplexing and filtering index hopped reads.

If you have biological replicates it is better to prep individual libraries for each. You can always pool reads later if required.

Controls and IP samples should require similar number of reads and input quantity but it is better to check with your analyst.
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Old 10-06-2020, 12:28 AM   #3
rozita
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Thank you for your reply!

Unfortunately I have already ordered the Multiplex Oligos Set1 and they have 12 unique sequences, so I can have a pool of 12 different libraries?
Is it going to be less reliable / any drawbacks that Iím unaware of to use this instead of the Dual Index ones?

For the biological repeats, yes of course I was going to prepare a different library for each of them. However, my question was that for each biological repeat, I have 6 samples so that is 6 libraries. To minimize any sort of variability during the processing of the sequencing samples, would it be better if when I pool the libraries prior to sequencing, that I keep the 2 biological replicates in separate pools or have them together in 1 pool?

Once again, thank you for your time and effort to reply!
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Old 10-06-2020, 03:03 AM   #4
nucacidhunter
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There is no drawbacks but pooling 12 libraries in one lane of S4 flow cell will output around 200M reads/library. If you are aiming for 30M reads/libray then SP flow cell (350-400M reads) will be more cost effective.

Sequencing will not introduce any major variation so you can sequence replicates in the same lane or separately.
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