Only phiX reads in my data

Question

[Picoskia]

Hi,

When i'm aligning my sequence data to my reference genome, i'm getting only a very small fraction of reads (less than 1%). I have checked a lot the data, and found that 99% of my sequences are corresponding to phiX genome.

What does it mean? Did my run went wrong?

Thanks.

[GenoMax]

Did you not do any QC on the prepped libraries? Like @Devon said below either your libraries failed or there was some other issue (if the library had passed QC).

[Picoskia]

unfortunately no......

[Devon Ryan]

A Qubit and a Bioanalyzer are your friends :)

Answer 1

[Devon Ryan]

Sounds like your library prep failed, so you ended up with all spike-in.

[Picoskia]

thank you for your answer, i should try to make a new library....

Answer 2

[harold.smith.tarheel]

You'll also see this result with high-quality libraries that have non-standard adapters (such as those requiring custom sequencing primers).

[Picoskia]

Thanks a lot, yes i have prepared a custom library with specific primers. Do you have any clue about why my reads are not mapping to my reference genome in this case?

[GenoMax]

If the sequencing center did not include your custom primers during the sequencing then the sequencing (of your sample) did not work (your sequence provider should repeat the sequencing at no cost to you, IF you had told them that you need a custom sequencing primer when you submitted the sample, otherwise you would need to pay to repeat).

Non-phiX reads that you have are probably sequencing artifacts and thus won't map to your reference.

[Picoskia]

i have give them the sequencing primers maybe my library went wrong

[GenoMax]

Can you ask them and confirm that custom primers were indeed used during run? It is not uncommon to forget this type of a thing since it is not required for "normal" runs.

If they did use the primer then the suspicion would shift back to bad libraries (provided your custom primers have been used before and proven to be good for other samples).