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Old 03-16-2019, 01:27 AM   #1
KB*
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Default HMW tail in the library. Remove or leave and sequence?

Hi there,
Skipping all the suffering.. I now have my libraries for ChIPs-Seq: good looking peaks, but also a high molecular weight something on the right of the upper marker Worth to notice, that only IPd samples have this peak, inputs do not have it.
PCR conditions:
- inputs ~2-2.5 ng, 12 cycles;
- IPs 0.5-1 ng, 15 cycles - all withing the recommended range in the kit.

Beads carryover? I have noticed that some beads are not sticking to the magnet. But I leave 2.5 uL behind not to aspirate these beads. Repeated treatment on the magnet did not help. I was thinking, may be my beads decomposed and there are tiny speckles I can not separate? I spinned some libraries 15 min 15000 rpm - does not help.

Second size-selection on some libraries diluted to ~100 pg/uL did not help either (may be DNA is too diluted for Ampure beads?)


I found this thread to be very useful,
http://seqanswers.com/forums/showthread.php?t=15224

but as it is quite old, may somebody else can tell me more on what that tail is and how "dangerous" it is for the sequencing? The sequencing facility proposes to do a trail with MiSeq and then if the libraries perform OK, the run on NovaSeq. Reasonable, but superexpensive. May be not worth it (more expensive than the new prep).

ssing did an interesting test. They denatured the samples and then checked them on gel - no HMW tail anymore! Hope to see that with my samples.. But still not getting what it is and how this tail can affect the sequencing.
Thank you all!
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Old 03-25-2019, 08:15 AM   #2
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In some of my early library preps, I also saw and worried about a high molecular weight smear before. I thought it was bead carryover, but it wasn't.

I think it is daisy-chained library resulting from depletion of primer or dNTPs. For more info, see:

1) the section titled "Primer Depletion and Library Over-amplification" in the Kapa Library Amplifcation Kit guide: https://www.kapabiosystems.com/docum...-kit-tds/?dl=1

2)https://ethanomics.wordpress.com/201...y-preparation/

3) http://seqanswers.com/forums/showpos...3&postcount=15

If that is correct, the libraries should be sequence-able, but the main thing is that your library quantification calculations (i.e. if nM based on the mean fragment length and Qubit concentration) might be inaccurate, and could affect the balance of reads if you are pooling libraries.

Does your facility run MiSeq Nano for a low price? That might be a good way to test the libraries / pooling balance.

Or you can run a qPCR-based library quantification kit (but I don't have experience with these) to get more accurate concentration measurements.

You can also try the suggestion of one of the links above of adding more primer, dNTPs, and enzyme to do one more round of PCR.
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Old 04-03-2019, 06:10 AM   #3
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Thank you @arctan for the answer,

I actually ended up doing one more PCR cycle. All but one library (input #15) looked OK for me. I tried to re-size-select #15 ending up with loosing DNA. Anyway, the samples were submitted, not sequenced yet. The sequencing facility sent me a very confusing QC file saying that all libraries failed The image is attached. For example, library #21 looks absolutely Ok for me. I am trying to get the answer what was the exact reason for failing. May be that was just a concentration issue. The plan for now is to prepare the new #15 and may be to remove small fragments from some libraries.

Any suggestions?
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Old 04-03-2019, 09:22 AM   #4
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Piggybacking off this a bit...I came here to post a very similar issue. I am seeing weird behavior at the tail of my run near the high MW marker. These libraries are post-spri bead cleanup (and my magnet/tubes don't seem to work as well as I have seen in the past), so am thinking I have some residual bead carryover. Two examples shown. Thoughts?
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Old 04-03-2019, 06:19 PM   #5
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The attached images are too small for me to read the axis units. However, the libraries seem to look perfectly fine for me.


Quote:
Originally Posted by KB* View Post
Thank you @arctan for the answer,

I actually ended up doing one more PCR cycle. All but one library (input #15) looked OK for me. I tried to re-size-select #15 ending up with loosing DNA. Anyway, the samples were submitted, not sequenced yet. The sequencing facility sent me a very confusing QC file saying that all libraries failed The image is attached. For example, library #21 looks absolutely Ok for me. I am trying to get the answer what was the exact reason for failing. May be that was just a concentration issue. The plan for now is to prepare the new #15 and may be to remove small fragments from some libraries.

Any suggestions?
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Old 04-03-2019, 07:23 PM   #6
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Quote:
Originally Posted by bman View Post
Piggybacking off this a bit...I came here to post a very similar issue. I am seeing weird behavior at the tail of my run near the high MW marker. These libraries are post-spri bead cleanup (and my magnet/tubes don't seem to work as well as I have seen in the past), so am thinking I have some residual bead carryover. Two examples shown. Thoughts?
This is ATAC-seq?
The proportion of longer fragments is relatively small. In our experience libraries like these can be sequenced without issues.
I do not believe the magnets are to blame - likely some cells targeted less than others?
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Old 04-03-2019, 10:06 PM   #7
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Hi KB*, I agree with luc (but I am not that experienced with this stuff), that the distribution of the library looks good.

I also cannot read the axes due to the low res images. I'm curious what your sequencing core says once you get a chance to talk to them.

Some of the libraries have a small peak (e.g. 16, 22, 23, 27) -- is this adapter-dimer contamination?

The estimated molarity of the libraries seem a little low (below 2 nM)? What concentration does your sequencing core require?
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Old 04-04-2019, 05:18 AM   #8
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@arctan, @luc, @bman
So happy to hear your answers and the discussion

Sorry for low res images. Prepared the new file with better images (only the seq.facility report. File is attached).

The seq. facility promised to "try to reply" tomorrow with explanations... waiting...

They asked 0.5 ng/uL, but I guess they meant 1.5 nM.

I am not sure what those "small fragments" (~60-70bp) are. primer-dimers? (Slide 36 in [2] ) or "artefacts" (p. 6 in [3] ) . Interestingly, in the manual of the kit I used for the libs prep they show an example of the successful library. That example also has the "small fragments", a slight peak tiling an even a bit of HMW DNA. So, that shall not be a problem.

Moreover, according to [2], Slide 37 either small peaks or wrong size libraries are sequencable.

Anyway, my QUESTION is whether I shall sequence these libraries (except #15)? Shall I remove the "small fragments"? (libraries 23, 27 in particular)?

[2] Slide 36
http://www.science.smith.edu/wp-cont..._June-2015.pdf

[3] https://www.agilent.com/cs/library/a...en-agilent.pdf
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Old 04-04-2019, 05:22 AM   #9
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If you are going to treat some samples to remove "small fragments" than the treatment may need to be applied to all samples. You don't want to introduce a bias.
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Old 04-04-2019, 05:37 AM   #10
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Quote:
Originally Posted by bman View Post
Piggybacking off this a bit...I came here to post a very similar issue. I am seeing weird behavior at the tail of my run near the high MW marker. These libraries are post-spri bead cleanup (and my magnet/tubes don't seem to work as well as I have seen in the past), so am thinking I have some residual bead carryover. Two examples shown. Thoughts?
Oh, @bman
I wish I know the answer. May be check the slide presentation I refer to in my previous answer [2]. I do not know may be you are preping some special kind of libraries. I am peping ChIP-seq libs and as you see my facility wants to see only one well defined peak at the expected size.

If you also expect to see only one peak and you can not remove HMW with the magnet, I think it is an overamplification artefact. This is what I did with my libraries; I denatured them at 95 oC for 5 min and cooled down at RT. When I run the sample on tapestation, I clearly saw ~600 bp peak indicating overamplification bubble. Another WORRYING thing also happened: the main peak (#300 bp) disappeared. My tech support said that my lib became ssDNA and that is why I do not see it. But I actually do not know...

Anyway in [2] slide 32 they recommend to check the libs like I described.

I can not really recommend anything as I do not have an experience in this, but many people say over-amplification it is not a problem for sequencing - please read the previous answers.

Just to add, when you have "strange" libraries at least here they do not give you any guarantees which means potential big money loss.

hope it helps

Last edited by KB*; 04-04-2019 at 06:05 AM.
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Old 04-04-2019, 07:25 AM   #11
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Quote:
Originally Posted by GenoMax View Post
If you are going to treat some samples to remove "small fragments" than the treatment may need to be applied to all samples. You don't want to introduce a bias.
Thank you @GenoMax for the suggestion. I guess with size-selection I can focus on libs 21-27 (libs 15-20 and 21-27 are prepared using different cell lines. So essentially those are the two different sets of samples). I think I will need to leave #16 as it is. For the best of my understanding the primer-dimer affects only the corresponding library, e.g. #16 in this case. Here #16 is one of the 3 replicas:

#15 - input, cell line 1;
#16 - IP1, cell line 1;
#18 - IP2, cell line 1;
#19 - IP3, cell line 1;
#20 - IP done with IgG, cell line 1

#21 - input, cell line 2;
#22 - IP1, cell line 2;
#23 - IP2, cell line 2;
#25 - IP3, cell line 2;
#27 - IP done with IgG, cell line 2

How about bias in this case?
I will need to prep the new #15. It will be exactly the same chromatin used for IPs 16,18,19,20. Unfortunately, I can not prepare new 16, 18, 19 and 20. I am not sure I have to replicate all the conditions., e.g. the number of PCR cycles and an additional PCR and additional size-selections I did. It is an input, I guess it just has to be a nice and as much as possible "complete" library? Am I right?

I still have some residues of previous library #15 (no additional PCR, please see the attached image). May be I can use that library? I will need to do some PCR to increase the DNA concentration. Note, during the last size-select I lost a lot of DNA.

I will appreciate any feedback on this
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Old 04-04-2019, 08:47 AM   #12
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Hi KB*,

Adapter dimers can be a significant concern since they cluster and bridge-amplify efficiently and their small molecular weight means that a minor peak (in mass) can correspond to a fairly molarity percentage of the library. The effect would be that you would get a higher number of undesirable reads in your sequencing. See: http://seqanswers.com/forums/showthread.php?t=22978.

Also see https://dnatech.genomecenter.ucdavis...ry-sequencing/ which says (for HighSeq 4000 / NovaSeq): "The new clustering chemistry is more sensitive to adapter dimers: a 5% adapter-dimer contamination can result in 60% of the reads coming from these dimers. Thus it is very important that there is no indication of an adapter-dimer peak (around 120 bp) on the Bioanalyzer trace."

HOWEVER, now that the images you attached are more high-resolution, the small peaks seem to be 65-80 bp, whereas usually adapter-dimers are around 120 bp. So those may be excess primer, which may cluster on the flow cell, but would not bridge-amplify).

If your small blips are indeed not adapter-dimers, then I think that your libraries look pretty good, so I am confused about your core's comments about the qc fail.

As to whether to sequence or not, part of the decision making relates to the sequencing depth needed and the budget. For example, if you are about to do a super expensive run and/or you need as much depth as possible, then it makes sense to be very careful. But if it's a smaller scale sequencing which doesn't squeeze your lab's budget, or if you would have so much extra depth that you don't actually need, then a smallish percentage of non-productive reads may be acceptable. Of course, we all wish for 100% perfection, but I'm trying to be pragmatic here.

For example, getting back the the large hump (daisy-chained). Those will probably fall apart once denatured, so they will sequence (see https://www.researchgate.net/post/Wh...a_cDNA_library). And if for some reason they were truly very large molecules, they would not cluster efficiently. So such a library would still sequence, although if you are pooling, your balancing of libraries might not be as accurate.

My institution's sequencing core offers an inexpensive MiSeq Nano with a fast turnaround time, so I've actually used that to do a quick run to make sure my pooled libraries will sequence well, and I use the MiSeq Nano results to alter the pooling balance if necessary for the larger scale, more expensive run.

(Someone correct me if I've posted anything inaccurate - apologies in advance)
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Old 04-08-2019, 05:31 AM   #13
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Dear all,

Dear @arctan @GenoMax, please help :-) I am lost.....

I got the answer from the sequencing facility. The libraries were failed because they have "short fragments". They are not primer-dimers or adapter-hexamers. They take the shortest fragment, subtract 120 and get this:
#15-152bp; #18-87bp; #19 -110bp etc.

They wrote: "The fragment size is less than 300bp, so there is small fragment in “remarks”.

I am not sure what they wanted to say

We were going to sequence (Novaseq) with paired reads of 150 bp. I guess that the core is trying to say that I have fragments shorter than the reads. Is that really a problem?

Last edited by KB*; 04-08-2019 at 05:43 AM.
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Old 04-08-2019, 06:06 AM   #14
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If your facility offers an option then do a MiSeq nano run to see what the data looks like in real life. That would give you some indication of whether you want to move forward or not on NovaSeq or if you should try to tweak pooling. If you don't have primer dimers (but have real short inserts) then take that into consideration when re-pooling.

Quote:
I guess that the core is trying to say that I have fragments shorter than the reads. Is that really a problem?
Yes that can be a problem. If you have very short inserts then with 150 bp reads sequencer will read into adapter at 3'-end at some point. This will cause Q-scores to drop and in general can be annoying. At least in your case not all samples are like this.

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Old 04-08-2019, 07:29 AM   #15
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Hi KB*, I agree with GenoMax. If you are going to do NovaSeq, that is a huge amount of money (at least to me it is). I've only done MiSeq and NextSeq with short reads, so I don't have experience with long read runs.

The figure and info in this post might be useful:
https://www.biostars.org/p/275387/
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Old 04-08-2019, 09:51 AM   #16
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Which sonicator did you use for your chromatin? Usually, it is very difficult to achieve such small inserts.

The seq facility is certainly correct that the size distribution is not optimal general purposes (small inserts generate redundant data). However, for ChIP-seq you only want to count and get the most precise mapping data possible. Shorter fragments give more precise data -- but only in case you can rule out sample degradation.


Quote:
Originally Posted by KB* View Post
Dear all,

Dear @arctan @GenoMax, please help :-) I am lost.....

I got the answer from the sequencing facility. The libraries were failed because they have "short fragments". They are not primer-dimers or adapter-hexamers. They take the shortest fragment, subtract 120 and get this:
#15-152bp; #18-87bp; #19 -110bp etc.

They wrote: "The fragment size is less than 300bp, so there is small fragment in “remarks”.

I am not sure what they wanted to say

We were going to sequence (Novaseq) with paired reads of 150 bp. I guess that the core is trying to say that I have fragments shorter than the reads. Is that really a problem?
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Old 04-09-2019, 03:29 AM   #17
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degraded lib2.jpg
Quote:
Originally Posted by luc View Post
Which sonicator did you use for your chromatin?
Usually, it is very difficult to achieve such small inserts.
Hi @luc,

Than you very much for your question about sonication. It is a deviation, but I am glad I can share my experience. I hope somebody might find it interesting/useful.

Either because our main sonicator gradually stopped working (Diagenode Bioruptor) or because I work with "difficult" cells (hematopoietic) or my samples were overcrosslinked (I used glycine stop crosslinking. I read that it is better to use Tris) - the sonication was failing to produce well fragmented chromatin. Moreover, the immunoprecipitation was always pulling down the longer fragments. I hypothesised that if I over-shear the chromatin I might be able to precipitate shorted fragments. Therefore, I switched to micococcal nuclease (MN). In the did not overdigest my chromatin (overdigested = only mono-nucleosomes), but I found out that with MN I can prepare larger batches of more concnetrated chromatin. Digested with MN niclei were "opened" by sonication on the probe sonicator Q sonica. It still was not easy to to break the nuclei! I believe probe sonication also shears some chromatin, but the small fragments are most probably the products of MN digestion.

Degradation
I think degradation will be visible on the electropherogram. Not sure about the core report. It is too flattened and no gel image. I believe the core is checking for the degradation.
I think I might caught one degrading library before. Look at the image - here we see multiple randomly sized fragments and the electrophoregrame is serrated. However, I do not know for sure.
Prepared libraries look different, so I hope there is no degradation.

Last edited by KB*; 04-09-2019 at 04:03 AM.
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Old 04-09-2019, 07:00 AM   #18
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Thank you very much, @arctan and @GenoMax for you help.

I now can only go Novaseq.

I got some more information from the core (below). Could anybody please comment?

1) The core fails libraries with fragments outside 200-400 bp range;
2) They say, "shorter fragments will not be sequenced with 150bp pair end read length, however, if you want to know the sequence of shorter fragments, we could provide you original data without demultiplexing, in that case, shorter fragments sequence will be included..."

Q1. Do I need data from the reads shorter then 150 bp? I know people are doing 50 bp reads. Shall I discard these reads?

Q2. What insert size I had to aim to?
I am using human derived cell lines. There is human reference genome. But... The cell lines were derived from the tumor tissues and also mutated during the cell lines derivation. It is known that the used cell lines have multiple chromosomal rearrangements, gene translocations, duplications, deletions etc. Therefore, it looks like the input DNA has to be sequenced without gaps as much as possible. Hence, 150 - 300 bp inserts (270-450 bp library fragments) are actually the best. Right now my libraryes have a half of fragments shorter then the lower optimal size, but there is also ~1/2 of the libraries within the optimal size. I understand that shorter fragments cluster more efficiently.

Q3. I have nothing to loose with my stock libraries. All libraries except #15 are safely stored in the core. Shall I try to tweak the library size, for example, size selecting with 0.7X (or similar working) "left size selection" [R]. The will lead to some loss of optimally sized library fragments. Does it worth it?
Will I be able to sequence all of the loaded libraries? If so, it does not matter if some reads are not informative.

Alternatively, I can do 0.95X Ampure beads removal of library fragments below ~100 bp. This shall be "safe" in sense of decreasing libraries complexity.

[R] https://ls.beckmancoulter.co.jp/file...SPRIselect.pdf
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Old 04-10-2019, 08:09 AM   #19
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Hi KB*,

thanks, the Nuclease treatment explains the shorter than usual fragments.
You really became creative with the protocol. I hope it works out.

I am not worried about a library degrading - we have never encountered such a problem. Your traces indicated that perhaps your sample was degrading before the prep - but the MNse treatment explains that.
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