1. An Overview of Applications for the MiSeq and HiSeq 2500 April 4, 2016 Kevin Shianna, Ph.D. Sequencing Specialist - Illumina, Inc. MGC USERS GROUP

2. The name of this group is now officially the MGC Users Group Our LISTSERV address is changed to MGC-USERS-L Our website address is changed to http://mgc.nichd.nih.gov

3. Steven L. Coon MGC Users Group meeting March 7, 2016

4. Outline: Amplicon sequencing What is meant? How large are your amplicons? Plasmid inserts When would you do it? Scale Coverage Cost What do you give to MGC? Primers to use Turn around time Targeted exome sequencing A special case of amplicon sequencing Combining gene lists

5. What is meant by amplicon sequencing? Can sequence essentially any type of DNA PCR amplicons Fragments of any size DNA Plasmid insert, or whole plasmids

6. When should you choose NGS to sequence your amplicons? or When does NGS make more sense than Sanger sequencing? Labor Cost

7. For most cases, we will be sequencing a set of amplicons Can multiplex, in parallel Rather than many individual PCR reactions If done as part of kit, then no individual purification steps Can easily design probes using Illumina’s DesignStudio website Example of 7, 14 & 37 genes http://www.illumina.com/designstudio http://www.illumina.com/designstudio

8. Illumina DesignStudio amplicon design output

9. Illumina DesignStudio amplicon order info

11. *prices are total cost for the number of samples; kits are for groups of 96 samples *fixed cost for library construction per sample is $9.10 *cost for running a FlowCell (v2/500 cycle) on the MiSeq is $1,100 *will use 1 FlowCell for up to 96 samples COST OF TRUSEQ AMPLICON KIT ALONE # of samples7 genes14 genes37 genes 965,5106,0616,917 1929,93810,82611,728 38416,13717,49618,436 TOTAL COST OF SEQUENCING (v2/500) # of samples7 genes14 genes37 genes 967,4848,0358,891 19213,88514,77315,675 38424,03125,39026,330 SEQUENCING COST PER SAMPLE (v2/500) # of samples7 genes14 genes37 genes 96788493 192727782 384636669 What are the numbers of amplicons sequenced? Cost calculations

12. # targets# amplicons% coverage 7 genes4982100 14 genes9113599 37 genes33244997 How to compare the costs of NGS vs Sanger? Should compare per amplicon (target) SEQUENCING COST PER TARGET PER SAMPLE (v2/500) # of samples7 genes14 genes37 genes 961.590.920.28 1921.480.850.25 3841.280.730.21

13. Coverage calculations for amplicon sequencing on MiSeq For # samples:96 Total bpStd MicroNano # genestargetedv3/150v3/600v2/300v2/500v2/300 v2/500 Gb >>3.8154.57.51.20.30.5 719,5801,1324,4691,3412,23435889149 1431,5207032,7768331,3882225693 37101,650218861258430691729 Assumes: % on-target:70 % pass filters and aligned:80 What average coverage can you expect? Generally, for targeted sequencing will aim for an average of 100x. This will ensure that almost every base is covered 10-20 fold

14. How many amplicons can be sequenced per run? Can be adapted to scale of project Depends on size of amplicons

15. Costs of sequencing kits Scalable Cost of sequencing per base drops dramatically

16. What is the turn around time? for sample sequencing 1-2 days for library construction 1-2 days for sequencing for data processing? 2-3 days for alignment and output Output will be the equivalent of a.vcf file Tells you what differs from expected Need to provide target sequences

17. When does it make sense to combine lists between investigators? TOTAL COST OF SEQUENCING (v2/500) # of samples7 genes14 genes37 genes 967,4848,0358,891 19213,88514,77315,675 38424,03125,39026,330 If you have a small number of samples and a small number of genes. For example: Dr. A has 48 samples, each with 7 genes Dr. B has 48 samples, each with 7 genes If run alone, each would pay $7,484. If designed and run together, each would pay $8,035/2 = $4,018 If they each had 96 samples, each with 7 genes, then they each would pay $14,773/2 = $7,387

18. What are costs for other types of amplicon sequencing? Only library construction costs listed here Need to add sequencing cost, depending on scale Custom PCR products $10/sample if primers are modified with adaptor $30/sample if using standard primers Plasmids Depending on size of insert, will probably sequence the entire plasmid $30/sample

19. Summary Sequencing amplicons using NGS can be a good alternative to Sanger sequencing if the scale is large enough. Can save both time and money. PCR amplicons and targeted exome sequencing are both on the continuum of amplicon sequencing. Rapid turn-around time I have provided templates for calculating coverage and sequencing costs. May make sense to combine amplicon sequencing with another investigator.

20. Website: mgc.nichd.nih.gov List serv: MGC-USERS-L Email: NICHDDIRMolGenomicsLab@mail.nih.govMolGenomicsLab@mail.nih.gov Phone: 301-402-4563 Walk-in: Bldg 10/Rm 9D41