Adding adaptors on DNA samples

[Elisa]

Hello,

I have done a Crispr/cas9 screen with my durg, and now I'm preparing the DNA samples. I'm confused that if I need to add a flowcell adaptors on both ends? I have inquired a platform that they told me I only need to add illumina sequence when I amplify the samples. And also barcodes on both ends or only sing end?
For my understanding, the primer should be in this order:
A flowcell attachment sequence+barcode+illumina sequence+target binding site.
Is that right?

Thank you!

[Ben3]

Hello Elisa can you tell us a little more about which sequencer you will be running your samples on? And do you plan to do a single- or pair-end sequencing run?

[Elisa]

Hello Elisa can you tell us a little more about which sequencer you will be running your samples on? And do you plan to do a single- or pair-end sequencing run?

Hello, thank you Ben3. It seems that they will use the DNBSEQ PE100. What bother me most is that do I need two indexs or one is OK to do a paired-end sequencing?

[Ben3]

Elisa I haven't personally run a DNBSEQ sequencer before, but since it's a pair-end run, I would say that you do need two different indexes (one on each side). This is part of Complete Genomics instruments and you can learn more about that process through this link. It explains how the entire sequencing process works for your sequencer and should help answer your question.

[Elisa]

Elisa I haven't personally run a DNBSEQ sequencer before, but since it's a pair-end run, I would say that you do need two different indexes (one on each side). This is part of Complete Genomics instruments and you can learn more about that process through this link. It explains how the entire sequencing process works for your sequencer and should help answer your question.

That's very helpful for me, thank you!😀

[Diannagellar]

Hello! I can help clarify your confusion regarding the use of flowcell adaptors, barcodes, and Illumina sequences in your DNA sample preparation for your CRISPR/Cas9 screen.

When preparing DNA samples for Illumina sequencing, you typically need to add flowcell adaptors and barcodes (also known as indices) to your DNA fragments. The flowcell adaptors are required for attaching the DNA fragments to the sequencing flowcell, while barcodes are used to identify individual samples when multiplexing multiple samples in a single sequencing run.

The primer design you mentioned is generally correct, but let's break it down step by step:

1. Flowcell attachment sequence: This is the sequence that allows your DNA fragments to bind to the Illumina flowcell. It is typically added during library preparation, usually through adapter ligation. The specific sequence depends on the Illumina platform you are using, so make sure to follow the manufacturer's instructions or established protocols.
2. Barcode (index): Barcodes or indices are short DNA sequences that are unique to each sample. They allow you to multiplex multiple samples in a single sequencing run, meaning you can sequence several samples together and distinguish them based on their individual barcodes. Barcodes are typically added during library preparation, either through adapter ligation or during PCR amplification. You can use single-end barcoding (where the barcode is added to only one end) or dual-index barcoding (where different barcodes are added to both ends of your DNA fragments). The choice between single-end or dual-index barcoding depends on your experimental needs and the sequencing platform you are using.
3. Illumina sequence: The Illumina sequencing platform requires specific sequences that allow the sequencer to initiate and read the DNA fragments. These sequences are usually added during PCR amplification, typically using primers that contain the necessary Illumina-specific sequences. The exact sequences may vary depending on the specific Illumina platform and chemistry you are using, so be sure to consult the manufacturer's guidelines or established protocols.
4. Target binding site: This refers to the sequence that is specific to your target of interest, such as the region you have designed your CRISPR/Cas9 screen against. The target binding site should be incorporated into your PCR primers to amplify the desired DNA fragments for sequencing.

To summarize, your primer design should include the flowcell attachment sequence, barcode (index), Illumina sequence, and target binding site. The specific order and locations of these sequences may vary depending on the library preparation protocol you are following, so it's important to consult the manufacturer's guidelines or established protocols for your specific sequencing platform.

I hope this helps!