Amplicon)sequencing)using)next gen)technology) - … · Amplicon)sequencing)using)next gen)technology) STAMPS)course)2013) Hilary) ... 5,525) 15.50%) SS_WWTP_1_25_11_ACGCA) 214,950)

Amplicon sequencing using next-‐gen technology

STAMPS course 2013 Hilary Morrison

& Sharon Grim, Joe Vineis

Overview •  Tag sequencing •  Primer design •  Amplicon sequencing on two Illumina plaIorms –  fusion primers – opKmizaKon – piIalls – current protocols

WHAT YOU WILL NOT HEAR ABOUT

RETIRED

EXCHANGED

Targets

•  Ribosomal RNA (SSU, LSU) •  Internal transcribed spacers (ITS)—fungi •  FuncKonal genes – nirS – butyrate kinase, butyrate transferase – others

Which variable regions to target?

V1 V3 V7 V8 V9

8F

V2 V4 V5 V6

V6 (967F-‐1046R) 60 bp V4 (518F-‐806R) 288 bp V4V5 (518F-‐1064R) 550 bp V9 (1380/1389-‐1513) for eukaryotes

341 518 806 926 967 1064 1513 1380

ITS (fungi)

18S

ITS1 ~350-‐400 bp ITS2 ~250-‐300 ITS1-‐ITS2 ~550-‐800

ITS1F ITS1R/ITS2F ITS2R

ITS1 ITS2 28S 5.8S

AmplificaKon process •  DOs:

–  High quality DNA template: e.g. 260/280 ~2 –  Replicate reacKons using high-‐fidelity polymerase –  NegaKve controls –  Pool, clean, size-‐select

•  Qiagen MinElute (FLX; Illumina v6), •  Ampure (Titanium; Illumina v4v5); •  Pippin Prep (Illumina pools)

–  QuanKtaKon •  e.g. PicoGreen, qPCR

–  VisualizaKon •  Bioanalyzer, Caliper, gel

•  DON’Ts: –  MDA or nested amplificaKon

Pre-‐Ampure Post-‐Ampure

What can go wrong? (sources of error) •  ContaminaKon •  PCR error (enzyme misincorporaKon) •  Chimeras •  PCR ectopic amplificaKon (non-‐rRNA) •  MulKple Sequence Alignment •  Distance measurements (gaps, etc.) •  Clustering methods

Post sequencing •  Basecalling •  QC pipeline •  Taxonomy assignment or clustering – QIIME – GAST (Sue Huse, 8/4) – mothur –  RDP Pipeline (hjp://rdp.cme.msu.edu/) – Oligotyping –  basic BLAST

•  VAMPS

16S-‐SPECIFIC PRIMER DESIGN

Design primer suite for conserved sites

•  Start with known primers •  Check matches against SILVA or other rRNA database – Phyla missed? – ProporKon missed?

•  Add variants or degenerate sites to expand coverage

Literature review

Database sources

3,194,778 Total 739,633 SSURef

Example: bacterial-‐archaeal universal primer v. 1

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

90.0%

100.0%

0 mismatches 1 mismatch 2 mismatches 3 mismatches

Bacteria

Archaea

Eukarya

GGACTAC[ACG][CG]GGGTATCTAAT

Example: bacterial-‐archaeal universal primer v.2

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

90.0%

100.0%


Bacteria

Archaea

Eukarya

GGACTAC[ACG][CG]GGGTATCTAAT GGACTAC[ACT][ACG]GGGT[AT]TCTAAT

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

90.0%

100.0%


Bacteria

Archaea

Eukarya

V6 primers 967F is pool of 4 oligos with 2-‐16X degeneracy

CNACGCGAAGAACCTTANC CAACGCGMARAACCTTACC ATACGCGARGAACCTTACC CTAACCGANGAACCTYACC

1046R: 4 oligos or 1 oligo with 16X degeneracy CGACAGCCATGCANCACCT CGACAACCATGCANCACCT CGACGGCCATGCANCACCT CGACGACCATGCANCACCT

CGACRRCCATGCANCACCT

V6 Primer Sequence Target RefDB exact match

967F1-‐PP CNACGCGAAGAACCTTANC Bacteria 63.3%

967F-‐AQ CTAACCGANGAACCTYACC Deferribacteres, Aquificales 0.1%

967F-‐U12 CAACGCGMARAACCTTACC AddiKonal Proteobacteria 14.0%

967F-‐U3 ATACGCGARGAACCTTACC AddiKonal Bacteroides; Spirochaetes 12.7%

1046R CGACRRCCATGCANCACCT Bacteria 95.4%

Keep level of degeneracy low (2-‐16X) Are rare, but important groups missed? Primers with 1-‐2 mismatches open work—pay ajenKon to Tm

V4V5 Primer Sequence Target RefDB exact match

518F CCAGCAGCYGCGGTAAN Bacteria 97.0%

926R1 CCGTCAATTCNTTTRAGT Bacteria 96.2%

926R3 CCGTCAATTTCTTTGAGT Verrucomicrobia,

Delta proteobacteria, AcKnobacteria

2.3%

926R4 CCGTCTATTCCTTTGANT Epsilon proteobacteria 1.3%

Why not use CCGTCWATTYNTTTRANT?

128 variants and only 40 match a known bacterial sequence!

Fusion primer modificaKons •  Phosphorothioate linkages •  Barcoding: 5-‐12 nt •  Indexing: 6-‐12 nt •  Fusion with plaIorm-‐specific sequencing primer sequences (vs. ligaKon)

•  Illumina fusion primers include bridge adapter regions

ILLUMINA TAG SEQUENCING

Illumina amplicon strategy

•  Fused primer approach, not adapter ligaKon – Primers much longer than 454 versions –  Include bridge adapter sequences – Minimize cost of barcoded primer sets

•  Many labs now have Knight 515F/806R sets (V4; >1000 indices) •  CombinaKon of 12 indices x 8 inline barcodes creates 96-‐plex set for other targets

Illumina amplicon targets

•  How long are the reads? – Hiseq: 100/150 PE – Miseq 250 PE and longer

•  How good is the quality towards the end? – Undetected errors will inflate alpha-‐diversity – Merged reads require overlap

100 nt PE HiSeq1000

V6R: 1046R

~60 bp complete overlap

967F

Read 1

Read 2

Longer reads on MiSeq

V5R: 926R

50-‐100 nt overlap

V4F: 518F

Read 1

Read 2

Start with v6

•  Original 454 pyrotag target-‐lots of experience •  Sequence overlap with v6v4 datasets •  Cheaper to do •  Greater sampling depth – 300-‐400 million clusters per lane possible – 3M reads at 96-‐plex – 300K reads at 960-‐plex

rRNA…TAATCTWTGGGVHCATCAGG-CCGACTGACTGA-TTGCGTGCGATC-TAGAGCATACGGCAGAAGACGAAC!

5' AATGATACGGCGACCACCGAGATCTACAC-TATGGTAATTGT-GTGCCAGCMGCCGCGGTAA…rRNA!

5’ adapter “pad/linker”*

*designed to have no homology to 16s or Illumina adapters/primers

“515F”

Knight lab sequencing primer 1

“806R-‐rc” “pad/linker-‐rc”* index-‐rc 3’ adapter-‐rc

Knight lab sequencing primer 2 RC is indexing sequencing primer

Fusion primer design

Learned during Miseq Training

•  First 4 bases of read 1 used for cluster finding (no change)

•  Next 8 bases (up through base 12) need to be high-‐complexity because of phasing/prephasing calculaKon

•  This is also true of read 2 •  Thus, need to have randomness in cycles 1-‐12 of both reads!

In-‐line bar codes vs. indices •  In-‐line bar codes cut into sequencing reads, but: •  Cluster finding requires random distribuKon of A,C,G,T at

start of read 1 •  We designed our primers to provide this diversity

–  5’ NNNN (1-‐4) –  Careful mix of bar codes (5-‐9)

•  Didn’t work –  Clusters are found, but amplicons are sKll ‘low complexity’ samples

–  Diversity needed at first 12 bases of both read 1 and read 2 •  Had to add high proporKon of PhiX or other ‘random’

library

5’ AATGATACGGCGACCACCGAGATCTACAC-‐ TCTTTCCCTACACGACGCTCTTCCGATCT-‐ NNNNTCAGC-‐ CNACGCGAAGAACCTTANC 3’

967F_PP_N4TCAGC: one of 4 primers with TCAGC barcode; forward read

Fusion primer design

5’ CAAGCAGAAGACGGCATACGAGAT-‐ CGTGAT-‐ GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-‐ CGACRRCCATGCANCACCT 3’

1046R_index_1 : single degenerate primer with index; reverse read

Bridge adapter Sequencing primer binding In-‐line barcode 16S PCR

Bridge adapter Index Sequencing primer binding (x2) 16S PCR

Similar for v4v5 suite

Primer synthesis •  >80 nt long •  PurificaKon needed –  DesalKng only –  PAGE (used for first set; expensive) –  No other opKon because of oligo length

•  Pooled 4 967F primers for each barcode (V6) •  Pooled 3 926R primers for each index (V4V5) •  Final set of 8 forward/barcoded and 12 reverse/indexed primers = 96 unique combinaKons for each domain

•  Working stocks @10 uM each

ILLUMINA SEQUENCING

Effect of low-‐complexity libraries

•  Clusters found correctly •  Then read hits highly

conserved sequence •  Quality scores reduced •  MiKgate with lower cluster

density and HC library spike-‐in –  over 50% PhiX on early Miseq runs

•  Illumina has sopware fix for problem on Miseq –  now ~2% PhiX on Miseq runs

Control gDNA: known community

Error rate

•  Generated data from HMP mock community gDNA •  Expected sequences recovered •  Aligned unique sequences to reference •  Sequencing errors, not quality scores

• Overall error: 0.52%

How does Illumina tag sequencing compare to pyrotag sequencing?

Analysis of v6 data from real samples

Sandra Mclellan samples: Illumina (HiSeq) overrepresents AcKnomyces; misses Arcobacter almost enKrely

Samples are: 3 HiSeq v6 amplicon datasets and 2 454 datasets (v6v4 and v6 only)

Does length of primers bias recovery?

Test alternaKves in 2-‐step PCR. – 16s primers only, followed by full-‐length fusion – 16s plus part of Illumina adapters, followed by adapters to bridge

Sample, condidons Barcode Index

SS_WWTP_1_25_11, 16s only 20 cycles GAGAC 7: GATCTG/CAGATC

SS_WWTP_4_11_11, 16s only 20 cycles ACGCA 8: TCAAGT/ACTTGA

SS_WWTP_1_25_11, split primers TCAGC 1:CGTGAT

SS_WWTP_4_11_11, split primers TCAGC 2:ACATCG

“Split” primers

967F_PP+seq CACGACGCTCTTCCGATCTACGTTCAGCCNACGCGAAGAACCTTANC

967F_AQ+seq CACGACGCTCTTCCGATCTCGTATCAGCCTAACCGANGAACCTYACC

967F_U2+seq CACGACGCTCTTCCGATCTGTACTCAGCCAACGCGMARAACCTTACC

967F_U3+seq CACGACGCTCTTCCGATCTTACGTCAGCATACGCGARGAACCTTACC

ACGTbridge+seq AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACGT

CGTAbridge+seq AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTA

GTACbridge+seq AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGTAC

TACGbridge+seq AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTACG

1046R+seq GGAGTTCAGACGTGTGCTCTTCCGATCTCGACRRCCATGCANCACCT

BridgeIndex_1 CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCC

BridgeIndex_2 CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGCTCTTCC

PCR condiKons Standard 3 x 33 ul amplicon reacKon plus negaKve control 1st round: either 16s only primers or 16s+parKal adapter Only 20 cycles (vs. 30) Pooled 3 reacKons Diluted 10 ul to 500 (1:50) Used 1 ul diluKon in 2nd round of PCR with 2nd set of primers, 5 cycles

PCR results

16s only, 20 cycles 16s only, +5 cycles 16s only, negaKve 16s only, 20 cycles 16s only, +5 cycles 16s only, negaKve Split, 20 cycles Split, +5 cycles Split, negaKve Split, 20 cycles Split, +5 cycles Split, negaKve

Sequencing results

Searched for match in raw data, read 1, each dataset >gi|343201492 Arcobacter cibarius TGGACTTGACATAGTAAGAACTTTCTAGAGATAGATTGGTGTCTGCTTGCAGAAACTTATATAC

Sample, condidons Count read1 Count Arcobacter %

SS_WWTP_1_25_11_Bv6v4 35,612 5,525 15.50%

SS_WWTP_1_25_11_ACGCA 214,950 2,309 1.10%

SS_WWTP_1_25_11_GACTC 240,520 2,606 1.10%

SS_WWTP_1_25_11_GTATC 392,570 5,445 1.40%

SS_WWTP_1_25_11_16s 6,106,654 900,824 14.80%

SS_WWTP_1_25_11_split 12,782,547 1,558,392 12.20%

SS_WWTP_4_11_11_16s 5,779,659 824,789 14.30%

SS_WWTP_4_11_11_split 17,102,934 2,108,534 12.30%

0"

5"

10"

15"

20"

25"

30"

35"

40"

45"

Beijerinckiaceae"

Alicyclobacillaceae"

Helicobacteraceae"

Xiphinematobacteriaceae"

Crenotrichaceae"

Rhodobacteraceae"

Auran@monadaceae"

Acetobacteraceae"

Xanthobacteraceae"

Tracheophyta"

Erythrobacteraceae"

Enterococcaceae"

Lactobacillaceae"

Phyllobacteriaceae"

Neisseriaceae"

Coxiellaceae"

Sinobacteraceae"

Listeriaceae"

Clostridiaceae"

Chi@nophagaceae"

RickeJsiaceae"

Bacteroidaceae"

Williamsiaceae"

Staphylococcaceae"

Rhodocyclaceae"

Streptococcaceae"

Nocardiaceae"

Nocardioidaceae"

Burkholderiaceae"

Bdellovibrionaceae"

Methylophilaceae"

Family"I"

Bacillaceae"

Brucellaceae"

Cryomorphaceae"

Microbacteriaceae"

Planctomycetaceae"

Bradyrhizobiaceae"

Moraxellaceae"

Legionellaceae"

Hyphomicrobiaceae"

Methylobacteriaceae"

Unassigned"

Rhodospirillaceae"

Oxalobacteraceae"

Paenibacillaceae"

Alcaligenaceae"

Caulobacteraceae"

Verrucomicrobiaceae"

Comamonadaceae"

Xanthomonadaceae"

Sphingobacteriaceae"

Sphingomonadaceae"

Rhizobiaceae"

Enterobacteriaceae"

Pseudomonadaceae"

Cytophagaceae"

Flavobacteriaceae"

MTG$vs$v4v5$

M51.MTG.RC"

M51.MTG.UC"

M51.v4v5.1step"

M51.v4v5.2step"

0"

5"

10"

15"

20"

25"

30"

35"

Micromonosporaceae"

Xanthobacteraceae"

Streptomycetaceae"

Nitrosomonadaceae"

Hyphomonadaceae"

Pseudonocardiaceae"

Methylocystaceae"

Hydrogenophilaceae"

Micrococcaceae"

Rhodospirillaceae"

Rhodocyclaceae"

SAR116"

Beijerinckiaceae"

Unassigned"

PiscirickeFsiaceae"

Paenibacillaceae"

Patulibacteraceae"

Oxalobacteraceae"

Psychromonadaceae"

Bacillaceae"

Erythrobacteraceae"

Acetobacteraceae"

Nocardioidaceae"

Bartonellaceae"

Phyllobacteriaceae"

Hyphomicrobiaceae"

Francisellaceae"

Bradyrhizobiaceae"

Neisseriaceae"

Mycobacteriaceae"

Alteromonadaceae"

Methylobacteriaceae"

Brucellaceae"

Enterobacteriaceae"

Cytophagaceae"

Verrucomicrobiaceae"

Microbacteriaceae"

Caulobacteraceae"

Mycoplasmataceae"

Alcaligenaceae"

Rhizobiaceae"

Xanthomonadaceae"

Nocardiaceae"

Sphingomonadaceae"

Sphingobacteriaceae"

Flavobacteriaceae"

Comamonadaceae"

Moraxellaceae"

Pseudomonadaceae"

MTG$vs$v4v6$

50ng.8cyc.R2"%"

367.v4v6"%"

Other evidence for ‘missing’ taxa

Arcobacter

Epsilons Rikenella

Primer Tm at 1.5-‐3.6mM Mg2+

Concentradon in working mix (uM)

Specific targets?

518F 64 10 Universal 926R1 52 8 Rikenella 926R3 57 1 926R4 55 1 Epsilons

Bacterial v4v5

Thermocycling: 94˚C, 3:00 25 cycles of: [94˚C, 0:30; 55-‐60˚C, 0:45; 72˚C, 1:00] 72˚C, 2:00 4˚C, hold

454 v6v4 and MiSeq v4v5 datasets

Sewage 19_SS 1 step 2 step Bv6v4

20_SS Bv6v4 21_SS Bv6v4

Leech I_GS1_R2 Bv6v4 REA_HDF Bv6v4

OPTIMIZING PCR CONDITIONS

or, how Sharon spent her spring

CondiKons to try

•  Primer concentraKon – 0.2-‐0.4 uM

•  Magnesium concentraKon – 1.5mM – 3.67mM

•  Annealing temperature – Tm of primers suggest anywhere from 52˚C-‐60˚C

•  Keep primer composiKon and cycle number constant

Leech

Ladd

er

sewage

Ladd

er

REA_HDF

55˚C 57˚C 60˚C 55˚C 57˚C 60˚C 55˚C 57˚C 60˚C

0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3

A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B A B

Effect on amplificaNon of

Condidon Leech REA_HDF

Increase temperature Decrease Decrease

Increase primer Increase Increase

Increase magnesium Increase Increase

Annealing temperature, Primers, A: 1.5 mM MgSO4 B: 3.6 mM MgSO4 All NTC at 55˚C

19_SS

Ladd

er

20_SS

Ladd

er

21_SS

55˚C 57˚C 60˚C 55˚C 57˚C 60˚C 55˚C 57˚C 60˚C

0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3



Condidon 19_SS 20_SS 21_SS

Increase temperature Decrease Decrease Decrease

Increase primer Increase Increase Increase

Increase magnesium Increase Increase Increase


(+)/(-‐)

55˚C 57˚C

0.2 0.3 0.2 0.3

A B A B A B A B

Arco_1

Ladd

er

Arco_3

Ladd

er

209_5M

55˚C 57˚C 60˚C 55˚C 57˚C 60˚C 55˚C 57˚C 60˚C

0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3



Condidon Arco_1 Arco_3 209_5M

Increase temperature Decrease Decrease N/A

Increase primer No Effect Minor increase N/A

Increase magnesium Increase Increase N/A


(+)/(-‐)

55˚C 57˚C

0.2 0.3 0.2 0.3

A B A B A B A B

Archaeal v4v5 •  Try annealing temperature, primer concentraKon, magnesium

concentraKon •  Hold constant 1X HiFi Buffer, 0.1 U Taq/uL, template •  Use Av6 and Bv6 as posiKve control for template

Primer Tm at 1.5-‐3.6mM Mg2+

Concentradon in working mix (uM)

arc517F1 63 10 arc517F2 59 10 arc517F3 58 10 arc517F4 56 10 arc517F5 56 10 arc958R 57 10


Av4v5

Ladd

er2

Av6 Bv6*

55˚C 60˚C

NTC

55˚C 60˚C

NTC

60˚C

NTC

0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3 0.2 0.3

A B A B A B A B A B A B A B A B A B A B

Annealing temperature, Primers, MgSO4 A: 1.5 mM MgSO4 B: 3.6 mM MgSO4 1X HiFi Buffer, 0.1 U Taq/uL. All NTC at 0.2 uM each primer, 1.5 mM MgCl2, 55˚C


Condidon Av4v5 Av6 Bv6*

Increase temperature Decrease No change N/A

Increase primer Increase Increase Increase

Increase magnesium Increase Increase Increase


*In comparison, the usual cocktail has 1X HiFi Buffer 2 mM MgSO4 0.2 mM each dNTP 0.4 uM each primer 0.02 U/uL Taq Plenty of DNA In 33 uL total

Arcobacter

Epsilons Rikenella

Primer Tm at 1.5-‐3.6mM Mg2+ Specific targets?

518F 64 Universal 926R1 52 Rikenella 926R3 57 926R4 55 Epsilons

Component 454 Bv4v5 Bv6 Av6 Av4v5

X HiFi 1 1 1 1 1

mM MgSO4 3.67 2 2 1.5 1.5

mM dNTPs 0.2 0.2 0.2 0.2 0.2

U/uL Taq 0.1 0.1 0.02 0.1 0.1

uM each primer (set) 0.3 0.4 0.2 0.3 0.3 annealing temp 60 60 60 60 55

cycles 30 25 25 25 30

If you see your products amplify with less Taq... tell us your secrets.

2 mM Mg2+, 0.02 U/ul Taq, 0.2 uM primers

3.67 mM Mg2+, 0.1 U/ul Taq, 0.37 uM primers

Current protocols bac v6 arc v6 bac v4v5 arc v4v5

HiFi buffer 1X 1X 1X 1X MgSO4 2 mM 2 mM 3 mM 3 mM dNTPs 0.2 mM 0.2 mM 0.2 mM 0.2 mM

combined primers 0.2 uM 0.3 uM 0.3 uM 0.3 uM PlaKnum HiFi 4 units 10 units 10 units 10 units Template 5-‐20 ng 5-‐20 ng 5-‐20 ng 5-‐20 ng Volume 100 ul 100 ul 100 ul 100 ul

PCR 3min 94o 94o 94o 94o 30 sec 94o 94o 94o 94o 45 sec 60o 60o 57o 57o 1 min 72o 72o 72o 72o 2 min 72o 72o 72o 72o

Cycles, step 1 25 30 30 30

Pooling, clean up Qiagen MinElute

plate Qiagen MinElute

plate Ampure, 1:1 Ampure, 1:1

Step 2 4 ul step 1 eluate 0 0 0 Cycles, step 2 5 0 0 0 QuanKtaKon Picogreen Picogreen Picogreen Picogreen Size selecKon Pippin Prep Pippin Prep Ampure Ampure

Typical amplificaKon results

4900

1900 2900

100

1100

300

500 700

Archaeal v4v5 pooled, on Caliper GX Bacterial v6 pooled, on Caliper GX

Size selecKon

v6 amplicons (Pippin)

v4v5 amplicons (Ampure)

Illumina amplicon QC

•  Filter out reads based on: –  ChasKty filter –  Ns

•  Then ajempt to merge forward and reverse reads •  Keep only those that match perfectly over the region of overlap (v6) or have fewer than 3 mismatches (v4v5)

•  Create consensus using basecall with higher Qscore •  Trim away adapters, primers •  Product goes into usual GAST pipeline, but as unique sequences plus frequency info, not all reads

2012-‐13 amplicon sequencing goals

•  Complete 454 projects in progress √ •  ReKre GS-‐FLX √ •  ConKnue to use HS1000 for metagenomics, short reads, V6 √ •  Use 96+ mulKplexed V6 amplicons on HiSeq (paired end

perfect overlaps) √ •  Use mulKplexed V4 amplicons (no overlap) X •  Run V4-‐V5 amplicons on MiSeq (parKal overlap for QC and

merging) √ •  ReKre PGM √

Final comments •  Amplicon sequencing is moving target

–  Not as well supported by next-‐gen companies as shotgun sequencing –  Databases growing –  Sample sources expanding (animal microbiomes, extreme environments, low

biomass targets, marine/freshwater/sediments/rock surfaces) –  Read lengths increasing; paired end reads possible –  Processing s/w –many approaches to filtering, trimming

•  So— –  Look at your raw data –  Assess expected coverage by primer sets –  OpKmize amplicon protocols and processing –  Evaluate accuracy, not just quality (run controls) –  Don’t hesitate to discard LQ reads –  Expect technological advances

Documents

Amplicon)sequencing)using)next gen)technology) - … · Amplicon)sequencing)using)next gen)technology) STAMPS)course)2013) Hilary) ... 5,525) 15.50%) SS_WWTP_1_25_11_ACGCA) 214,950)