5' RACE question


#1

Hello, just a quick question I hope someone can help me with,

If you do 5’ RACE on B-cell mRNA, do you see a leader sequence on the 5’ end of the variable region? If so, how long is it, roughly?

Many thanks

William


#2

Yes, typically ~150 nucleotides


#3

@caschramm Thanks for the quick reply! I hadn’t thought through the large impact this has on overall sequence length :frowning:


#4

It’s meant we can’t sequence on MiSeq any more, but we’ve had good results with 2x300 on HiSeq2500 in rapid run mode…


#5

Well, I think you may be able to sequencing on MiSeq if you can prepare two libraries. Then combine the sequencing results of the two libraries together using some bioinformatic approaches. Well, this just the thoughts.


#6

If you are careful to place your reverse primer right at the beginning of the constant exon you might get away with 5’ RACE with a 2 x 300 bp V3 MiSeq. You can also get another 10 bp by programming the run for 305 cycles each direction (t’s not much but it can be useful).
While some 5’UTRs can be 150 bp, most are less - and the average seems to vary in different species (we’ve noticed that primates seem to have longer 5’UTRS compared to rodents).


#7

Good point about species differences -I work almost exclusively with human samples, so I forget that sometimes.

We have not had any success with 2x300 on MiSeq in at least two years, including a test of the newest kits just a couple of weeks ago (although we will be retesting that shortly). The quality is too poor to cover an amplicon of more than 450-500 bp. With the 500-550 bp amplicon from 5’ RACE (of human BCR), we get low rates of merging R1/R2 and high error in the overlap region even for those reads which can be merged. We are already using primers close to the 5’ end of CH1 (~25 nt in, if I remember correctly).


#8

There was a major issue with the V3 2 x 300 bp kit from the end of 2015 until late last year. I’ve heard various explanations from Illumina as to the underlying issue (the current claim is that there is a metabolite that is building up to levels that affect the R2 read quality but only in a proportion of kits) but they claim it is now resolved.
We have definitely seen an improvement in quality for the more recent V3 kits so maybe they are telling the truth this time.
At the moment we do use RACE for gene discovery - mainly for non human samples - but have switched to multiplex PCR for individualized databases as this allows us to avoid the long UTR issues.


#9

Assuming that you are referring to the 500 cycles Rapid SBS v2 kits: Did they give you are better quality in the covered sequence parts than the 2x300 MiSeq runs? Are you using them in an asymmetric setup (300+200)?


#10

We are interested in the Ig subtype, and are planning to try the approach of Schanz et al, who use 250x250 bp reads to cover the variable region, and an index read to capture the subtype discriminator. Would have liked to use 5’ race, but the need to include the leader makes things look a little too tight, even with 300x300 reads.