Plasmid library representation and diversity

[Indumati Sharma]

Hi All,

We recently purchased the Brie library from Addgene and sequenced the library subsequent to transformation and PCR amplification. 

I wanted to know what the typical threshold is to judge if we have got enough of representation and diversity of each of the sgRNA. I see from Joung et al (2017)- 

"An ideal sgRNA library should have >70% perfectly matching guides, <0.5% undetected guides, and a skew ratio of less than 10." 

Along these terms:

1) I was wondering what skew ratio is? 

2) We see many sgRNAs which have reads less than 100. Is that concerning?

3) We sequenced 4 technical replicates, out of which two have shown to have reasonable representation of sgRNA , whereas the other two replicates do not. I am at crossroads right now to decide if I need to repeat plasmid library amplification. If we do what factors determine where we would need to restart the process? Do we start from library transformation step? or Can we go back to the extracted plasmid DNA and re-seqence to see if it was just sampling problem.

Your help is much appreciated.

Thanks,

Indu

[Julia Joung]

Hi Indu,

To answer your questions:

1. Skew ratio refers to the number of NGS reads for the 90th percentile divided by those of the 10th percentile.

2. Depends on how many NGS reads you had total. The QC metrics are for a sequencing depth of 100 reads/sgRNA in the library, and you should have <0.5% guides that were not detected (i.e., 0 reads).

3. I usually pool technical replicates together before sequencing, but you can also do this after sequencing. If you followed the protocol to amplify the sgRNA library for NGS QC, then you should not have to repeat this step. Usually the problem occurs at the library transformation step, but of course this depends on what your library metrics currently look like.

Best,

Julia

--
You received this message because you are subscribed to the Google Groups "Genome Engineering using CRISPR/Cas Systems" group.
To unsubscribe from this group and stop receiving emails from it, send an email to crispr+un...@googlegroups.com.
To post to this group, send email to cri...@googlegroups.com.
For more options, visit https://groups.google.com/d/optout.

[Indumati Sharma]

Thanks alot for your help.

Cheers,

Indu

On Tuesday, 26 March 2019 10:59:57 UTC+13, Julia Joung wrote:

Hi Indu,

To answer your questions:

1. Skew ratio refers to the number of NGS reads for the 90th percentile divided by those of the 10th percentile.

2. Depends on how many NGS reads you had total. The QC metrics are for a sequencing depth of 100 reads/sgRNA in the library, and you should have <0.5% guides that were not detected (i.e., 0 reads).

3. I usually pool technical replicates together before sequencing, but you can also do this after sequencing. If you followed the protocol to amplify the sgRNA library for NGS QC, then you should not have to repeat this step. Usually the problem occurs at the library transformation step, but of course this depends on what your library metrics currently look like.

Best,

Julia

On Thu, Mar 21, 2019 at 8:52 PM Indumati Sharma <math...@gmail.com> wrote:

Hi All,

We recently purchased the Brie library from Addgene and sequenced the library subsequent to transformation and PCR amplification. 

I wanted to know what the typical threshold is to judge if we have got enough of representation and diversity of each of the sgRNA. I see from Joung et al (2017)- 

"An ideal sgRNA library should have >70% perfectly matching guides, <0.5% undetected guides, and a skew ratio of less than 10." 

Along these terms:

1) I was wondering what skew ratio is? 

2) We see many sgRNAs which have reads less than 100. Is that concerning?

3) We sequenced 4 technical replicates, out of which two have shown to have reasonable representation of sgRNA , whereas the other two replicates do not. I am at crossroads right now to decide if I need to repeat plasmid library amplification. If we do what factors determine where we would need to restart the process? Do we start from library transformation step? or Can we go back to the extracted plasmid DNA and re-seqence to see if it was just sampling problem.

Your help is much appreciated.

Thanks,

Indu

--
You received this message because you are subscribed to the Google Groups "Genome Engineering using CRISPR/Cas Systems" group.

To unsubscribe from this group and stop receiving emails from it, send an email to cri...@googlegroups.com.

[Vahid]

Hi Julia, 

May I please know your opinion about our NGS results for the library representation?

We bought CRISPR V2 Libraries A and B from Addgene and amplified it based on the protocol in Nature paper. Here is the summary of our NGS results comparing amplified libraries to the originals:

 

	A -  Amplified	A -  Original	B -  Amplified	B -  Original
Number of reads processed	25,419,745	28,134,930	32,923,233	26,168,525
Number of perfect guide matches	18,008,956	19,844,560	22,417,200	17,788,394
Number of nonperfect guide matches	4,798,152	5,374,973	6,754,415	5,383,355
Number of reads where key was not found	2,612,637	2,915,397	3,751,618	2,996,776
Percentage of guides that matched perfectly	79.0	78.7	76.8	76.8
Percentage of undetected guides	1.7	0.4	3.3	2.3
Skew ratio of top 10% to bottom 10%	13.24	10.42	20.66	18.06

First of all, I know that this is not the ideal result based on the criteria mentioned in the Nature protocol (Less than 0.5% undetected guides, and a skew ration of less than 10). But another concern is that why our Original Library B is not optimal?!! What could be the problem? We followed all the steps based on the protocol. I would really appreciate it if you help me with this. 

Thank you for your time, 

Vahid 

On Tuesday, 26 March 2019 08:29:57 UTC+10:30, Julia Joung wrote:

Hi Indu,

To answer your questions:

1. Skew ratio refers to the number of NGS reads for the 90th percentile divided by those of the 10th percentile.

2. Depends on how many NGS reads you had total. The QC metrics are for a sequencing depth of 100 reads/sgRNA in the library, and you should have <0.5% guides that were not detected (i.e., 0 reads).

3. I usually pool technical replicates together before sequencing, but you can also do this after sequencing. If you followed the protocol to amplify the sgRNA library for NGS QC, then you should not have to repeat this step. Usually the problem occurs at the library transformation step, but of course this depends on what your library metrics currently look like.

Best,

Julia

On Thu, Mar 21, 2019 at 8:52 PM Indumati Sharma <math...@gmail.com> wrote:

Hi All,

We recently purchased the Brie library from Addgene and sequenced the library subsequent to transformation and PCR amplification. 

I wanted to know what the typical threshold is to judge if we have got enough of representation and diversity of each of the sgRNA. I see from Joung et al (2017)- 

"An ideal sgRNA library should have >70% perfectly matching guides, <0.5% undetected guides, and a skew ratio of less than 10." 

Along these terms:

1) I was wondering what skew ratio is? 

2) We see many sgRNAs which have reads less than 100. Is that concerning?

3) We sequenced 4 technical replicates, out of which two have shown to have reasonable representation of sgRNA , whereas the other two replicates do not. I am at crossroads right now to decide if I need to repeat plasmid library amplification. If we do what factors determine where we would need to restart the process? Do we start from library transformation step? or Can we go back to the extracted plasmid DNA and re-seqence to see if it was just sampling problem.

Your help is much appreciated.

Thanks,

Indu

--
You received this message because you are subscribed to the Google Groups "Genome Engineering using CRISPR/Cas Systems" group.

To unsubscribe from this group and stop receiving emails from it, send an email to cri...@googlegroups.com.

[Julia Joung]

Hi Vahid,

Your amplified results actually look pretty similar to the original library, so in your case the problem is not in the amplification procedure, but rather the library itself. Unfortunately, not all of the libraries available on Addgene were cloned using the methods we outlined in our screening Nat Protocols paper, so they do not pass our QC metrics. The mouse GeCKO library has a similar issue.

These libraries are not ideal, but it does not necessarily mean that the screen will not work. I would just recommend to increase your coverage, if possible, to account for the extra noise.

Best,

Julia

To unsubscribe from this group and stop receiving emails from it, send an email to crispr+un...@googlegroups.com.

To post to this group, send email to cri...@googlegroups.com.

To view this discussion on the web visit https://groups.google.com/d/msgid/crispr/ce1611da-2a44-4997-b1b8-ea1877a6911b%40googlegroups.com.

[Vahid Atashgaran]

Hi Julia, 

Thank you for your quick response. In that case, increasing the cell coverage from 500 to 650 would suffice? that is 30% increase!   

Kind regards,

Vahid 

[Julia Joung]

Hi Vahid,

The coverage that may be necessary varies depending on the screen, so I would recommend trying 650 first and see what your screen looks like.

Best,

Julia