Fungal ITS sequencing Illumina

[richrr]

Hi,

We recently did Fungal ITS paired-end sequencing using Illumina. It turns out that overall, the read 1s do not, whereas read 2s pass the sequence quality metrics. Before sequencing we had checked the quality of the DNA by running a gel, a nanodrop.and qubit. Can someone give us some hints as to why this might have happened?
Also, is it ok to use only the read 2s from this sequencing run and analyze it as a single end data?

Thanks for your help.

[Brian Bushnell]

Could you post the quality histogram and base-composition histograms for read 1 and read 2? There are any number of reasons, such as laser failure during one run and not the other, or end-specific adapter contamination. Also, what are your sequence quality metrics? They might simply be too strict.

[richrr]

I will try to post the images soon. The quality metrics are the default Q>30 imposed by the Miseq software.

[richrr]

Sorry for the late reply.

[Brian Bushnell]

Wow, that looks like a colossal failure of the sequencing machine. Maybe something went wrong with the reagent.

You certainly could just use read 2 and treat it as single-end.

[jennomics]

Did you figure out an explanation for this? What primers did you use? I just got back my first MiSeq ITS run, and it had the same issue. I haven't spent anytime troubleshooting yet - just came across this and figured I'd ask. Thanks!

[richrr]

Turns out that we needed better primers. We switched to a different protocol (suggested by Illumina) and a different set of primers from a paper (I'll post the citation soon). We should have the results soon. Will let you know if it worked.


Using only read 2 as a single end read is not much use. I am currently using the paired-reads that passed QC.

[d00b]

hi Richrr, you switched to a different protocol which is suggested by illumina. do have any information on the new protocol? any website link for the protocol? Thanks

[richrr]

The 16S protocol using nextera adapters, replace the 16S primers with ITS specific primers.

[Teagtech]

ITS sequencing using the 16S protocol

I am currently trying to prepare samples for ITS sequencing using the Illumina 16S protocol. I ordered the ITS primers with the Illumina overhang sequences attached and have been trying to optimise the PCR conditions. However, I seem to be generating multiple bands. I'm a bit new to this and could use some help.

Thanks :-)

[nucacidhunter]

There are a few primers for ITS amplification in literature. You may first try primers without any overhang to optimise PCR reactions. Presence of multiple bands depend on the sample composition because ITS region length varies among species.

[Teagtech]

Thank you for replying so quickly :-)

[ruchi1st2002]

Hi
Use a touch-down PCR protocol and at last step keep for 10min in 75 C then to 4degree forever..