Deep sequencing question

[frank]

Using the lentiguide-puro (2plasmid gecko system), if I use the forward primer (hU6), how long should I read before I get the complete sequence of the sgRNA. The PCR amplicon is roughly about 400bps. So is it necessary to read the full length? Any comments would be much appreciated.

Thanks.

[Neville Sanjana]

Hi Frank,

Apologies for not answering this when you emailed me earlier today!

We typically use 80 cycles with our Illumina primers (see http://genome-engineering.org/gecko for our primers). I know other folks have used custom primer designs and gotten away with even less cycles. For MiSeq, we typically buy the 150 cycle kit but only do a 80 bp forward read (and also an index read to get the sample barcode on the reverse primer). The reason is that only the first 20bp of the sgRNA is variable (i.e. the U6-proximal portion of the sgRNA). The rest of the sgRNA sequence will be the same for all constructs in the library.

Hope that helps,

Neville

On Thu, Mar 12, 2015 at 10:03 PM, frank <franco.vi...@gmail.com> wrote:

Using the lentiguide-puro (2plasmid gecko system), if I use the forward primer (hU6), how long should I read before I get the complete sequence of the sgRNA. The PCR amplicon is roughly about 400bps. So is it necessary to read the full length? Any comments would be much appreciated.

Thanks.

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[Thomas Leete]

Neville,

First of all, this group is incredibly useful and I'm grateful for how superbly your whole system is documented between this group, your supplementary methods, and the protocols.

I came to this thread while confirming how long the forward sequencing read needs to be.  I was hoping that the answer was that you only read 50bp, (in other words 10bp into the 20bp sgrna) but alas, I guess we'll have to do 100bp reads.

Your reply to Frank leaves me with a related question.

Why do you read from the reverse primer at all?   In your July 2014 Gecko primer spreadsheet, the oligos have indexes on both ends.   As it is, I've ordered 4 forward and 4 reverse for sequencing the plasmid library but don't know why I couldn't have just used a single reverse primer since we will be reading through the index anyway with the forward Illumina primer.   What is the point of having different reverse primers, why would you care about the reverse index?

I've already sent samples to our Core so hopefully I'm not wrong.

Thanks,

Thomas

[Neville Sanjana]

Hi Thomas, 

These are great questions! It is very important to understand the functions of the reverse and forward primers for PCR#2. Both reverse and forward primers add Illumina adaptors and unique barcodes. In addition, the forward primers add stagger (to increase amplicon diversity and avoid monotemplate issues that cause problems for Illumina sequencing). 

There are many possible primer designs that will work as long as 1) they avoid monotemplate and 2) they include a unique sample identification scheme (i.e. unique R barcode, unique F barcode, or unique combination of F and R barcodes).

We use the reverse primer to get the reverse index (in Illumina parlance, the "I1 read"). We never sequence the reverse read itself ("R2 read"), as we get the sgRNA sequence off the forward read ("R1 read"). If you're just sequencing 1 plasmid library, you can mix a few forward primers (purpose: avoid monotemplate since forward primers contain stagger) and use 1 reverse primer. If you're sequencing 2 plasmid libraries together, then you can mix forward primers (again, stagger) and use one reverse primers for library 1 and a second reverse primer for library 2. The reverse barcode, captured in the I1 read, allows you to demultiplex the run and assign reads to library 1 or library 2.

Hope that helps,

Neville

[Thomas Leete]

Thanks Neville,
That is helpful for sure.  I basically know what I'm doing insofar as Illumina Deep Sequencing.  You had just mentioned in your response to Frank that you read the I1 index and this puzzled me since there was an inline index that you must also have read.  I guess the point is to have ways to combine the various F and R primers to make lots of combinations for deep multiplexing, which is a cute idea.   

For the plasmid pools I'm using 4 forward primers with unique in-line barcodes and staggers, one for each of 4 different plasmid pools (two copies of your guidepuro A and B) to be combined in one lane.  I am only going to read from R1 (100bp) and will see the bar code in-line before the sgrna sequence. I will not sequence I1 or R2 at all. 

So, for the plasmid pool, I think that this will work fine.  If I was interested in reinventing the wheel, I think that I might redesign them by  taking out the in-line barcode in the forward primer as well as a couple base pair from the PCR annealing site which combined will let me get away with a 50bp read instead of 100 and would then also read I1 for the index.   This is a little more cost effective but I'll probably stick with what I've already bought.

Thanks again,

Thomas

[Neville Sanjana]

Hi Thomas,

Sounds good. As you recognize, there are many primer strategies that will all work fine. :) And it's a great idea to sequence some PCR replicates (as you are doing) to measure any technical variability. Although it might not be necessary, I should also mention that we usually spike in a small amount of PhiX (5-10%) to increase diversity but it might be fine to omit this given the staggers.

Good luck with the readout!

Neville

[Sara Bird]

Hi Neville,

Thank you so much for all your help! I know we've gone over this a lot, but as further clarification of the primers posted on your website, can we leave off the stagger sequence for the reverse primers as long as our forward primers contain stagger? Thank you in advance!

Best,

Sara

[Neville Sanjana]

Hi Sara,

I haven't personally tried this but I think it should work fine. If you give it a try, please let us know how it goes!

Best,

Neville

[Vikas Daggubati]

Hi all, 

I've done a couple rounds of deep sequencing with great results thus far. I've tried the method in which I barcode my experiments with the forward primer and use the same reverse primer for all the samples. In addition, adding Ns to your stagger instead of a defined stagger further adds complexity to your sequencing. That said, I've never added PhiX to my sequencing and it appears as though it runs well. I hope that helps. 

Best, 

Vikas

[Rudolph]

Dear all,
can i ask you for some suggestions?
we are having problems with ours MiSeq sequences, we are having less reads, only 4 million per run and we don't understand why.

we do the first PCR, take 5 ul, use it to run the second pcr: for pool A we use the combination F1 to F4/ R1 +R6 and for Pool B F5 to F8/ R2 +R5.
run on gel, cut the band, purify with qiagen kit, a further purification with Ampure beads, check the DNA  with the bioanalyzer: one pick only at the right size and then proceed.

we run it 3 times:
first time 3.5 mil reads, we though about a mistake in DNA concentration,
second time: load the sample more concentrated, we got even less reads

then we clone the pcr purified product in topo ta vector (after A-tailing) and sequence 96 clones just to see if we have some other DNA contamination:
all clones had a sgRNA inside (i can track barcode and target gene)

order new oligos, repeat PCR with a new sample run for the
third time: 4mil reads!

any suggestion??

Thanks a lot in advance,
best,
Rudolph

[Fiammetta Falcone]

Dear Neville,

I will use the NextSeq, do you think that 75cycle will be enough?

Thanks,

Fiammetta

[Shirley]

Hi Neville,

 

Our lab is sending Gecko library for sequencing. The company requested Illumina kit # and method when you constructed the library. Can you please advise? Thanks.

 

Shirley

On Thursday, March 12, 2015 at 10:42:51 PM UTC-4, Neville Sanjana wrote: