How many number of samples per lane

[smurugesan]

Hi everyone,

I am a new user to Illumina MI-SEQ, in a single lane how many 16S Amplicons we can mix or in other way, to sequence 1000 samples, how may times we have sequence. Can any one suggest the kits to sequence using 300x2 in Illumin? Thank you.

[rudzik79]

Illumina suggests 96 samples for their regular 600 v3 kit.

[smurugesan]

Great, Thank you. A list of customized forward primers with barcodes and Reverse primer without barcode, is it possible to sequence amplicon size of 530bp using 600 v3 kit?

[Jessica_L]

Technically, yes, but whether you would want to do so will depend partially on your coverage needs and your tolerance for sequencing errors.

Using paired end reads of 300bp from either end will cover the amplicon with about 70bp of overlap in the middle, but reads in only one direction covering ~230bp on either side of that. You may not have a lot of coverage on the amplicon ends to indicate whether a detected variant is real or an error. Whether that would be a problem for you will depend, at least partly, on the design of your experiment.

[Brian Bushnell]

I personally do not think sequencing 530bp inserts on Illumina 2x300bp kits is a good idea, as those kits have shown very low quality and very poor consistency in the past, meaning that a mere 70bp overlap risks a high chance of complete failure or simply inaccurate results. It makes more sense to design experiments around a methodology that is known to be robust. That means either 2x250bp or insert sizes with much more overlap so as to be tolerant of low-quality tails. "Low quality" here can mean 100% incorrect (or N) tails which makes the data unusable.

[smurugesan]

Thank you Jessica and Brian for your valuable suggestions. In Illumina guide, they suggested to sequence an amplicon of 550bp or smaller. So I thought it would be ideal, and actually We are planning to sequence V1-V3 regions. In addition, I would like to ask your expertism. I am new to this technology, By using barcodes only in forward primers and common reverse primer for all the sample, Is it possible to distinguish the sequences read through reverse primer to do overlapping? I am not clear with it. If anybody explain this part, it would be great. Thank you.

[Brian Bushnell]

If you only have barcodes on one end, you need 96 unique barcodes if you 96-way multiplex. However, due to "barcode-hopping" it is ideal to design experiments with unique barcodes on each end (96 unique forward, 96 unique reverse) when cross-contamination/crosstalk is problematic (which depends on your experiment). If cross-contamination is not important 96 unique pairs are commonly achieved by 12 unique barcodes on one end and 8 unique barcodes on the other.

However, as for your question - "Is it possible to distinguish the sequences read through reverse primer to do overlapping?" - I'm not really sure what you're asking, but read merging software takes care of read-through and does not need specific barcodes.

[smurugesan]

Thanks for your explanataion Brian. Its really helpful. For e.g I have 96 different barcodes in my forward primers, But reverse primer is without barcode, My question is if illumina sequence in both directions. We have barcode sequences only in forward, how do we distinguish the output through reverse primer for each sample to do the merging with output through forward primers? Sorry for my ignorance, just i didn't get the concept clearly. Thank you.

[nucacidhunter]

I assume that your forward primers have inline 5’ sample specific barcode. You will have to ligate adapters or have a partial adapter overhang to add adapter sequences by PCR. In Illumina sequencing every Read1 will have a corresponding Read2 (they are normally marked as N1 and N2 in sequence header) so demultiplexing by forward primer barcode is fine. Though you will have to use a custom script to assign reads to each sample.

[smurugesan]

Thank you so much @nucacidhunter. That clears my doubt, thanks again.