Sequencing

Details about how to sequence the amplicons

Sequencing libraries are now ready to run on an Illumina sequencer.

We have been sequencing student samples on the Illumina miSeq in the UAF Genomics Core lab.

We use Paired End 300bp reads (PE300) to make sure that read 1 and read 2 of ~500bp fragments overlap.

It is important to split a run with another library or load a lot of phiX. This is because the ITS2 primers, and a long stretch of ribosomal sequence where the primers are located are identical in all of the amplicons. Without another library in the mix, or phiX, the computer apparently merges clusters with identical sequences in the beginning of the read. This results in artificially low quality scores for the sequences.

Even for a large class, ~10% of a miSeq flow cell should give you plenty of data.

We haven't tried it, but you may also be able to use Illumina PE250bp reads (250bp paired end reads), and use a higher throughput machine, such as the HiSeq or NovaSeq. Some sequencing companies charge quite low prices for ready to run samples on a fraction of a flow cell (~$100), such as Psomagen [formerly Macrogen], and DNA Link).

Adaptors

Our sequencing adaptors (PCR2 primers) are dual indexed iTru adaptors developed by Travis Glenn and colleagues, and detailed on their web page, and in their publication. These are the same as the Illumina truSeq adaptors (except for the indices), and add the same i5 and i7 sequencing regions as the Illumina truSeq adaptors.

Alternatively, you can also use the truSeq adaptors from Illumina.