300-500 range is the optimal size
600 cycle sequencing is only available on the MiSeq On the NovaSeq the maximum can be 500 cycles on the S-prime
Clusters: group of DNA positioned closely together, each cluster represents thousands of copies of the same DNA strand in a 1-2 micron spot. The spot is about a micron. You need to have the dilution of thelibrary such that they can evenly space the single stranded DNA library
MiSeq is 1000 DNA stand cluster Next seq is a 5000
Patterned Flow cell has a higher cluster density
Shut down the instrument from the software and then shut down the instrument from the switch.
How long do you keep the 1N NaOH before you consider it expired. Use new bottles/smallest bottle possible. 1N NoOH solutions are not stable enough. Personally not keep it more than 6 months.
Frozen aliquots: Still thorw them out after 6 months.
Paired-end reading When Illumina started, it could only do 36 bases and that was a knowledge to align them. But when you have information about the both ends you have a better chance of good alignment.
Index seq is a separate process Another limitation of sequencing is longer you sequence, lower the quality of your sequence
Experiment Manager/Local Run Manager/Base Space/
You can use Base Space for run monitoring. Proactive us a component of Basespace.
Combinatorial Dual Indexes Combinatorial Unique Dual Indexes
.bcl file are the primary data that comes out of the sequencer. Its one for every tile that is sequenced.
*RTA2 and RTA3
RunInfo.xml, images and this will result in the data files.
Color normalization is done in the first 26 cycles
As we progress throught the cycles, the signals would drop. It used to compensate by amplifying the intensity. An example: there was a loss of signal and the machine was calling this as a G
Cluster location are .clocs Cluster Intensities are .cif
Base calling and quality score are .bcl
Phasing needs to be <0.4%. This happens when a library in the cluster will have a base being read that is shorter on the sequence Prephasing will be sequencing the read that is next to the majority of them being read.
These could happen alot with expired reagents. Could happen due to worng washing, tween 20 concentration/contamination Do temporal lines washing. On Miseq do it once a month. Use bleach.
On four color chemistry, the base with the highest intensity is called.
Pass filter
IT measures the intenisty of the highest signal and then
Clusters passing filters are those one that remain after the calculation of pass filter.
On the pattern flow cell there is a possibility of having, in addition to having polyclonal cluster
Quality Scores: estimate of the probabliity of error in base calling Quality model:
Q30: what fraction of the reads should be better than Q30 (1 in 1000 error or 99.9% accuracy)
Percent aligned and error rate Spiking PhiX % Aligned is the percent of clusters in which the first 25 cycles align to the PhiX reference genome Error rate is the rate of mis-matches between sequencing data and PhiX reference genome
For low complexity libraries, spiking PhiX is recommended so that there is mixed material
So lower the complexity, higher the spike-in of the PhiX
BaseSpace Sequence Hub Local Run Manager
Have a reference guide for any instrument that you use handy. It can change quite frequently. Miseq: once every few moths, NovaSeq: quicker
Data output: 65-3000Gb
There are four flow cells available.
SP: 0.4Tb S1: 0.5Tb S2: 1.25Tb S4: 3Tb
Keep the used S4 flow cells handy. They can help with the wash
When you are transitioning to NovaSeq or a new platform what kind of determination would you make Sequence Coverage Calculator: to help not sequence any more than you need to and what kit to use
Standard: Run one library pool across the XP: Run separate library in each lane
Cluster Cartridge Library Tube ExAmp Reagents SBS Cartridge Flow Cell SP Manifold Buffer Cartridge
Dock , Lane Kit
Denature and Neutralize Library Pooled input library 1-3nM Final concentration of 250pM on S4 Flow Cell
Take out the flow cell and wipe it with isopronanol wipes and then wipe the flow cell with lens paper. After when the flow cell is placed on the instrument, take a can of air and blow it all over.
Make sure you clean the solution out of the MiSeq by washing it with water thoroughly. Dry it, shake it, then dry it again.
Make sure the gaskets are also positioned well in MiSeq flow cell before putting it in the instrument.
Illumina has started to move away from bcl2fastq and is now using bclconvert
Use this to understand the