First SQK MAP 006 experiment24 Sep 2015
We’ve just finally found the time to break open the new SQK-MAP-006 kits from Oxford Nanopore. These kits are notable because they introduce the first really major changes to the chemistry for some time.
- First up, the speed has been doubled from ~30 bp/s to ~75 bp/s. The assumption is this will increase yields, but it will be interesting to see what - if any - effect it has on quality profile. The worry would be that increased speeds would increase the chance of missing events (transitions between signal levels), which would manifest as deletions after basecalling.
- Secondly, the previous hairpin-motor complex (which enabled 2D reads and also stalled the complement strand) has been jettisoned to return to a simpler setup. As I understand it, the hairpin remains (and is now biotinylated and pulled down by beads to ensure very high 2D yields) but the second motor has gone. The new motor I assume is clever enough to be able to stall both the template and complement strand. It will be interesting to compare translocation times of the two strands (in SQK-MAP-005 the complement strand went through the pore more slowly, as it was retarded by two enzymes).
The new chemistry is accompanied with a new Metrichor basecaller workflow specific to SQK-MAP-006.
A notable change, looking at the returned FAST5 files, is that the model is now considering signal levels from each of the 4^6 possible combinations of 6-mers when doing basecalling. Before 5-mers were used. Does this mean that the ionic flux through the nanopore is in fact affected by 6 or more bases, rather than the 5 that we initially assumed? Or was 5 simply chosen to simplify the analysis. If the latter - and this seems likely - this may help with basecalling accuracy and it will be interesting to see if it resolves any previously difficult to sequence motifs (we looked at such under represented sequences in our recent paper here in the context of 5-mers: http://www.nature.com/nmeth/journal/v12/n8/full/nmeth.3444.html)
It does not seem to be supported to call older, pre-SQK-MAP-006 data with the new 6-mer model basecaller.
Links to FAST5 data files
So far we have done four SQK-MAP-006 runs. Two were generated with natural DNA, and two were generated with the low-input library that includes a PCR step.
Each of the files below are archives of the runs following base calling with Metrichor. We also provide a subset of one of the runs in ‘raw’ format which has the individual signal measurements (i.e. before event detection is carried out).
The raw files are available via the ENA FTP site
|Run||Basecalled data||2D pass FASTA|
|MAP-006-2||Basecalled and raw||Pass FASTA|
|MAP006-PCR-2||Basecalled and raw||Pass FASTA|
Head over to Jared Simpson’s blog to see some early results of using these data for assembly polishing.
As always, thanks to Josh Quick for his masterful library preparation technique.