PacBio services at CGS

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ChengWei WU

CGS, HKU

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http://www.pacb.com/applications/

Whole Genome Sequencing— Multiplexed WGS— Structural variants study (by PCR amplification, data analysis in progress)

Target Sequencing— Multiplexed amplicon sequencing

Complex Populations— Full-length 16S sequencing— Shotgun metagenomics (project in progress)

RNA Sequencing— Iso-Seq (data analysis in progress)

Epigenetics— Not yet start [ 4-methylcytosine (4-mC) and 6-methyladenine (6-mA) ]

http://sysbio.gzzoc.com/methsmrt/

→ √

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3http://www.pacb.com/wp-content/uploads/2015/09/Revolutionize-Genomics-with-SMRT-Sequencing.pdf

Sequencing output of PacBio Sequel system on instrument brochure:

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0

1

2

3

4

5

6

7

8

9

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Raw sequencing data output (Gb)

— average 5.3 Gb per cell

Cell #

[Gb]

Sequencing output of PacBio Sequel system at CGS:

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

16,000

18,000

20,000

Average polymerase read length (bp)

— average 13,272 bp per cell

Cell #

[bp]

0

100,000

200,000

300,000

400,000

500,000

600,000

700,000

800,000

Estimated raw read (as P1 value)

— average 447k reads per cell

Cell #

[Read#]

Polymerase read length (bp) of the latest 4 cells

20 kb

20 kb20 kb

20 kb

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Overview of CGS PacBio service workflow:

Project discussion with CGS

Sample QC

Sample ready for submission

Service confirmed by returning signed quote

Quotation preparation

Data collection or Bioinformatics service

Project sequencing report

Sequel SMRT cell sequencing

SMRTbell library construction

Library QC

Updates for the project progress

Sequencing run QC

Publication(Acknowledging CGS support in papers)

With CGS full-support

User CGS

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DNA Library Type QuantityConc.

(ng/μL)Comments

gDNALong insert (≥ 20 kb) with BluePippin size selection

15 μg >100 Gel-photo shows no signs of DNA degradation

gDNAMultiplexed library pool (10 kb protocol) with/without BluePippin size selection

10 μg(in total)

>50

(1) Gel-photo shows no signs of DNA degradation(2) Multiplexing 5 Mb microbial genomes up to

12-plex per cell with complete genomes assemblies (<10 contigs)

Completed-Amplicons

(1) Long insert without multiplexing(2) Multiplexed library pool (up to 6 kb - 7 kb)

2.5 μg(in total)

>25

(1) Ready for library construction(2) Purified PCR product with gel-photo(3) Sample pooling is recommended for amplicons

of similar sizes as +/-15% of the mean size

Tagged-Amplicons

Multiplexed library pool (up to 6 kb - 7 kb)>20 ng(each)

>2

(1) Ready for 2nd round PCR as barcode-PCR(2) Purified PCR product with gel-photo(3) Sample pooling is recommended for amplicons

of similar sizes as +/-15% of the mean size(4) The requirement is also for full-length 16S

sequencing

Sample recommended quantity for submission:

RNA Library Type QuantityConc.

(ng/μL)Comments

Total RNAcDNA library for the full-Length + >1kb transcripts

2.5 μg >300 RIN value ≥9

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Service price reference:

PacBio ServiceService Price

(HKD)Per Sample Price

(HKD)Service Content

1 x WGS ─ with 20 kb protocol

$16,410 $16,410.0

─ 1 x 20kb library construction

─ 1 x Sequel SMRTcell sequencing

─ BluePippin size selection

─ Pulse field gel electrophoresis (for QC)

4 x WGS─ with multiplexed 10 kb protocol

$23,010 $5,752.5

─ 4 x 10kb library construction

─ 1 x Sequel SMRTcell sequencing

─ BluePippin size selection

─ Regular gel electrophoresis (for QC)

12 x Amplicon Sequencing─ with multiplexed amplicon protocol

─ amplicons size ≤ 6 or 7 kb

$18,030 $1,502.5─ 12 x barcoding PCR

─ 1 x library construction

─ 1 x Sequel SMRTcell sequencing

$2,541.44 USD = $19,823.23 HKD

30 Mb of genome with 100X coverage for 20kb library sequencing, service price browsed on 27-Nov-2017.

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A subsidy of $5,000 HKD for each eligible project.The quotas are limited,

contact us ASAP!!!

Subsidized experience-price is …

Thank you thirdgenseq.cgs@hku.hk or http://cgs.hku.hk/portal/