Advancing Your Research with RAD Sequencing. 4 key considerations for utilizing RAD-Seq effectively in genetics and genomics studies

Transcription

1 Advancing Yur Research with RAD Sequencing 4 key cnsideratins fr utilizing RAD-Seq effectively in genetics and genmics studies

2 Table f Cntents 3 Intrductin 4 Why RAD-Seq? 5 Cmmn Applicatins and Publicatins 6 Have a Plan 7 Input DNA is Key 8 Cverage = Quality 9 Knw Thy Biinfrmatics! 10 Fur Key Cnsideratins

3 Intrductin The arrival f next-generatin DNA sequencing (NGS) has heralded in a new age f genmics, where large scale genme sequencing prjects are nw accessible t cnsrtia and individual investigatrs alike. Created frm sme f the lessns learned frm the genmics team at Flragenex, this bk will infrm yu: Why RAD sequencing is a NGS prtcl yu need t use Hw RAD can be leveraged acrss a variety f scientific applicatins Cnsideratins fr planning prjects The reasn why DNA quality is imprtant What sequencing cverage is ideal Biinfrmatics slutins available t analyze RAD data 3

4 Why RAD-Seq? Restrictin site Assciated DNA Sequencing (RAD-Seq) is fr life scientists wh need t assay genetic bimarkers in their research. At it s cre, RAD-Seq is a fractinal genmic sequencing strategy that enables identificatin and gentyping f genetic variatin using nextgeneratin DNA sequencing. Unlike ther array-based gentyping systems, RAD-Seq allws scientists t flexibly develp massive amunts f genetic infrmatin, fr a brad spectrum f mlecular genetic applicatins, with minimal up-frnt effrt. 4

5 Publicatins and Applicatins RAD-Seq has been used in ver 100 peer-reviewed scientific publicatins Marker Discvery Linkage Mapping Eclgical Genmics GWAS Phylgenetics Applied Genetics 5

6 Have a plan! Maximizing the results f yur RAD-Seq prject requires asking ne simple questin. What d I really want ut f this prject? Once the cre gal is established, mre technical questins shuld be addressed: Hw much data d I need? Hw shuld I handle RAD-Seq library prductin? Which next-generatin sequencing systems shuld I use? Hw will I analyze and publish the results? What additinal tls r knwledge d I need t accmplish the abve? Careful cnsideratin f these factrs and prper design f yur prject at inceptin is critical fr btaining impact results with RAD. 6 Measure twice, cut nce - Ancient prverb

7 Input DNA is Key Using high quality genmic DNA in yur prject is imprtant t help facilitate and simplify RAD-Seq library prductin. Our team at Flragenex believes that input DNA is an verlked cmpnent f many NGS prjects. A representative agarse gel image shwing the crrect amunt and quality f DNA fr RAD-Seq is shwn belw. 7 Adapted frm Flragenex Genmic DNA Input Requirements Frm

8 Cverage = Quality The key t generating cnsistent, high quality gentyping results with RAD-Seq is the depth f sequencing cverage. SNP calling versus sequence depth 100 At Flragenex, ur experience has taught us that 15x t 20x cverage prvides the ideal cmbinatin f rbust accuracy and minimized sequencing expenditure. Percentage f SNPs cvered with sequence reads x t 20x Sequencing Cverage is Optimum Average Read Depth (fld) 8 Chart adapted frm Bentley, et. al. Accurate whle human genme sequencing using reversible terminatr chemistry. Nature 2008

9 Knw thy Biinfrmatics! Data analysis is a critical cmpnent f any RAD-Seq prject using next generatin DNA sequencing. A variety f pen surce biinfrmatics tls are effective fr analysis f RAD-Seq data including: STACKS Bwtie GATK SAMtls Our analysis team at Flragenex uses Bwtie and SAMtls in cnjunctin with internal sftware fr analysis and reprting results t clients. 9

10 4 Key Cnsideratins 1. Plan Crrectly Set yur prject up fr success early 2. Use High Quality DNA It s abslutely essential fr RAD library preparatin 3. Next-Gen Sequencing Plan t generate 15x t 20x cverage fr impact results 4. Biinfrmatics Understand yur analysis needs and find the right sftware r slutin. Fr mre infrmatin frm the wrld leader in RAD-Seq visit: