Minion sequencing

rjlebbink · 10-26-2018, 04:26 AM

Hi all,

I have a very basic question. We have ±10 samples that we would like to sequence using MinION. These are PCR amplicons that we would like to multiplex in a single run.

Is it possible for me to attach index/adapters to these amplicons by a second PCR using custom primers that I can order as a standard primer from any primer company? If so, are there any recommendations for this? Can I just add the index right upstream of the gene-specific primers, or do I need to add an additional adapter up/downstream to allow sequencing of the amplicons via MinION? I am just trying to understand whether I need to use the nanopore PCR Barcoding Kit, or whether I can just add these (for much less $$ and effort) by PCR myself?

Cheers

gringer · 04-07-2019, 04:12 PM

Nanopore sequencing kits can be broken down into two basic categories:

1. Ligation chemistry
2. Rapid adapter chemistry

The ligation chemistry produces high yields and doesn't require any special sample preparation, but needs more external reagents (e.g. dA-tailing buffer, ligation buffer), and produces a few physically-chimeric reads (about 2-4% of reads). It's also the most similar to how Illumina sample prep happens.

The rapid adapter chemistry is extremely specific (I haven't seen any instances of chemistry-induced chimerism, and in-silico chimerism is usually less than 0.1%), but requires specially-prepared samples. ONT aren't telling customers their particular chemical modifications to get the rapid adapter click chemistry to work. They provide appropriately-modified primers in their PCR kits.

For PCR, the recommended process is to add anchor sequences to primers, and those anchor sequences are used in a two-step PCR to bind to the ONT-modified primers.

There's a whole bunch of other ways to do it. As one example, you could amplify up your products, then throw them into a rapid barcoding kit: 1 round of PCR with another ~10 mins of prep. The disadvantage of that approach is that you won't get full-length sequences; all sequences will be fragmented at the point of transposase binding.

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10-26-2018, 04:26 AM	#1
rjlebbink Junior Member Location: Europe Join Date: Apr 2014 Posts: 3	Minion sequencing Hi all, I have a very basic question. We have ±10 samples that we would like to sequence using MinION. These are PCR amplicons that we would like to multiplex in a single run. Is it possible for me to attach index/adapters to these amplicons by a second PCR using custom primers that I can order as a standard primer from any primer company? If so, are there any recommendations for this? Can I just add the index right upstream of the gene-specific primers, or do I need to add an additional adapter up/downstream to allow sequencing of the amplicons via MinION? I am just trying to understand whether I need to use the nanopore PCR Barcoding Kit, or whether I can just add these (for much less $$ and effort) by PCR myself? Cheers

04-07-2019, 04:12 PM	#2
gringer David Eccles (gringer) Location: Wellington, New Zealand Join Date: May 2011 Posts: 838	Nanopore sequencing kits can be broken down into two basic categories: 1. Ligation chemistry 2. Rapid adapter chemistry The ligation chemistry produces high yields and doesn't require any special sample preparation, but needs more external reagents (e.g. dA-tailing buffer, ligation buffer), and produces a few physically-chimeric reads (about 2-4% of reads). It's also the most similar to how Illumina sample prep happens. The rapid adapter chemistry is extremely specific (I haven't seen any instances of chemistry-induced chimerism, and in-silico chimerism is usually less than 0.1%), but requires specially-prepared samples. ONT aren't telling customers their particular chemical modifications to get the rapid adapter click chemistry to work. They provide appropriately-modified primers in their PCR kits. For PCR, the recommended process is to add anchor sequences to primers, and those anchor sequences are used in a two-step PCR to bind to the ONT-modified primers. There's a whole bunch of other ways to do it. As one example, you could amplify up your products, then throw them into a rapid barcoding kit: 1 round of PCR with another ~10 mins of prep. The disadvantage of that approach is that you won't get full-length sequences; all sequences will be fragmented at the point of transposase binding.