seq only part 1

Question 1

what are the different parts of an illumina library

Answer 1

p5 oligo

index 2 - i5

read 1 primer

insert

read 2 primer

index 1 - p7 - required

p7 oligo

Question 2

What is the sequential order of the reads that are generated from
an Illumina library?

Answer 2

read 1 primer

index 1 - i7

index 2 - i5

read 2 primer

Question 3

If a customer asked for 151-10-10-151 for their sequencing
arrangement, what does this mean?

Answer 3

151 - read 1 primer

10 - index 1 - i7

10 - index 2 - i5

151 - read 2 primer

Question 4

How long is the insert of a library?

Answer 4

no required length

we usually say 300-350bp but no requirement!

There is no “specific” requirement for an insert size, as the sample
input and/or library type. In an ideal world, the insert size should be
~300bp to 350bp to allow to be PE150 to be maximally used but
never a requirement.

Question 5

Are libraries generally
dsDNA or ssDNA? How are they loaded onto the flow cell
(dsDNA or ssDNA)?

Answer 5

they are prepared as dsDNA then denatured using NaOH and loaded as ssDNA

The workflow/product of most library preparation kits is dsDNA.

• There are some kits that produce ssDNA libraries, but these are
  not common.
• When the libraries arrive to the lab, they are QC’ed in their dsDNA
  state; however, prior to loading on the flow cell they will be
  denatured (ssDNA will bind/sequence on the flow cell) using a
  base, such NaOH.

Question 6

Q5: Can the insert be RNA or can it only be DNA?

Answer 6

only DNA
if RNA, converted to cDNA via reverse transcriptase

Only DNA, the sequencer cannot read RNA; if the “material” being
sequenced is RNA, it will be converted to cDNA using a reverse
transcriptase (RT)

Question 7

Q6: What are the 4 parts of Novogene’s library QC process?
Q7: What equipment is used for performing these 4 checks on the library?

Answer 7

volume - micropipette

concentration: qubit ng/ul

size: fragment analyzer

molarity: qPCR

Volume: [micropipette] minimum 10ul
* Concentration: [Qubit] – concentration provided in ng/ul
* Library Size: [Fragment Analyzer] - similar to a bioanalyzer or
tapestation; allows for accurate measurement of library size.
* Molarity: [QuantStudio] determine molarity using primers that
bind to the ends of the library (p5 and p7); allows for accurate
measurement of sequenceable material (Illumina library)

Question 8

Q8: Which one is more accurate – Qubit or qPCR? Why?

Answer 8

qPCR provides a more accurate quantification than Qubit because
it measures only sequenceable material through amplification. In
contrast, Qubit quantifies all double-stranded DNA, including nonsequenceable components such as dimers, primers, and partially
ligated adapters—molecules that contribute to the Qubit signal but
may lack the necessary P5/P7 adapter sequences for sequencing

Question 9

Q9: Do more clients perform Qubit quants or qPCR? Why?

Answer 9

Although qPCR is more accurate, most clients tend to use Qubit
because it is quicker/easier.
Qubit + Fragment analyzer can provide a molarity and would take <5
minutes to run both (and is generally more accessible). The qPCR
requires a specialized equipment + reagents and require a few
hours to run.

Question 11

What does it mean when the qPCR-nM is greater than the Qubit-derived nM?
What does it mean when the qPCR-nM is less than the Qubit-derived nM?
What does it mean when the qPCR-nM is equal to the Qubit-derived nM?

Answer 11

Qubit > qPCR: some library fragments may not have adapter
ligated.

• Qubit < qPCR: there might be library fragments that are singlestranded due to accidental or intentional denaturation. Our
  Qubit assay only measures double stranded DNA (due to the kit
  we use).
• Qubit ~ qPCR: sample present is mainly library

Question 12

Q13: Why is proper quantification crucial for the success of the sequencing run?

Answer 12

Based on the molarity (qPCR), we can make sure we load the “right” amount, based on what is loaded on the flow cell.

Question 13

Q14: What is included in the official library QC report?

Answer 13

Sample name

sample ID

Index seq

lib qubit

peak size

Lib vol

qPCR

peak desc.

lib QC results

Question 14

Q15: Why is a gel electrophoresis not as good for check the library size,
compared to using a Fragment Analyzer/Tapestation/Bioanalyzer?

Answer 14

Although a gel can work; it doesn’t give you the resolution you need to make accurate confirmations on library size.

Question 15

What is an important consideration when reviewing pass, hold,
or fail libraries for a client? Should clients be worried off-the-bat
when their libraries do not pass our QC?

Answer 15

Our library QC standard are not reflective of a specific library
type/starting material.

• They are generally based on a “picture” perfect library that should go on
  a sequencer; however, depending on the protocol/starting material
  material.
• A ‘pass’ does not imply the data will be perfect either, according to their
  library type and vice-versa.

Educate your clients, especially new ones, during the sales cycle:
1. Does the Fragment Analyzer profile match what you are expecting for your
library type?
2. Is there enough library to put on the sequencer to achieve the desired output?

Question 16

Is there a specific trace/distribution for a
library? If not, why not?

Answer 16

Novogene does not have library QC standards for each different
library type; also, the same library type could look a little different,
depending on the starting material.

• It is impossible to have standards for all the library types out there
  – we have rather “vague” standards for pass, hold, fail – so
  important to pay attention to the actual details in the library QC
  but do not downplay the output guarentee.

Question 17

Q18: What flow cell types does Novogene offer at this time?

Answer 17

On the NovaSeq X Plus, we offer PE150 (cycle 300 kits) on the 10B
and 25B; the 1.5B FC has all options available (consult your district
director for further evaluation).

Question 18

What sequencing strategy does Novogene primarily run? Why
does Novogene not run other sequencing strategies?

Answer 18

PE150
* As one of Illumina’s largest clients, Novogene is operating >80 Illumina
sequencers globally. As we need to stock reagents globally, we bulk
buy our reagents, where we get a discount from Illumina and pass on
the savings to you. As a result, running a PE150 FC with us, might be be
the cost of running shorter reads (i.e. PE100) with another
company/core.

• In addition, Novogene has established and maintained an efficient
  workflow for sequencing libraries. By having all libraries pass through
  the same read strategy/FC, we are able to load/start up our sequencers faster (you can imagine, by having different flow cell types and read
  lengths to sort through, this will slow down the lab in loading/starting
  up a sequencer.) This allows us to return data within a competitive TAT.

Question 19

Q20: What are adapters dimers? Are they problematic? If so, why?

Answer 19

These are two types of dimers we generally see: 60-80bp and 120-
160bp; the latter are more problematic as they contain all the
parts of the library without the biological sequence/insert.
* Smaller fragments cluster better
than larger fragments
* They should be cleaned-up;
if possible (NVG has purification