Unique, dual-matched adapters mitigate index hopping between NGS samples. Kristina Giorda, PhD

Transcription

1 Unique, dual-matched adapters mitigate index hopping between NGS samples Kristina Giorda, PhD 1

2 Outline NGS workflow and cross-talk Sources of sample cross-talk and mitigation strategies Adapter recommendations 2

3 NGS workflow 3

4 Library construction Fragmentation End repair and A-tailing Adapter ligation Bead cleanup Library amplification Bead cleanup Y-adapters: 13 bp are complementary Library conversion of top and bottom strand 4

5 Indexing strategies Ligation Library amplification 5

6 NGS target capture enrichment IDT xgen Lockdown Probes Individually synthesized Individual QC for every probe Individually normalized Pooled 6

7 Sequencing 7

8 What is sample cross-talk? Reads are assigned to the wrong sample 8

9 Applications that might be impacted by cross-talk Low-frequency somatic variant detection false positives from other samples Ancient DNA research a single sequence may support DNA survival or contamination Viral detection false positives from other samples Gene expression bleed over from one sample to another Microbial profiling bleed over from one sample to another Kircher M, Sawyer S, et al. (2012) Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. Nucleic Acids Research, 40(1):e3 e3. D Amore R, Ijaz UZ, et al. (2016) A comprehensive benchmarking study of protocols and sequencing platforms for 16s rrna community profiling. BMC Genomics, 17(1):55. 9

10 Custom dual indices AGBT 2014 IDT has been making custom dual indices for a long time 10

11 Sources of sample cross-talk Contamination Index hopping during multiplex capture Index hopping during cluster amplification Misread bases within index sequences Sample carryover from previous sequencing runs D Amore R, Ijaz UZ, et al. (2016) A comprehensive benchmarking study of protocols and sequencing platforms for 16s rrna community profiling. BMC Genomics, 17(1):55. 11

12 Sources of sample cross-talk Index contamination 12

13 Combinatorial indexing 13

14 Unique, dual matched indices reduce contamination misassignment P5 and P7 ligations are independent Index contamination may occur on any sequencing platform 14

15 Unique, dual-matched adapters reduce contamination misassignment exponentially 16 libraries were prepared and captured with the IDT xgen AML Cancer Panel Libraries were sequenced on the NextSeq System (Illumina) 0.09% of reads were filtered out that would have been misassigned with combinatorial indices 15

16 Sources of sample cross-talk Index contamination Index hopping Index swapping Index cross-talk Spreading of signal 16

17 Multiplexed target enrichment index hopping Target enrichment index hopping primarily occurs during post-capture PCR Index hopping may occur on the P5 and P7 side 17

18 Approach for measuring multiplex capture index hopping 16 libraries were prepared and 1-, 4-, 8-, or 16-plex captures were performed with the IDT xgen AML Cancer Panel as using 500 ng per library Libraries were sequenced on the NextSeq System (Illumina) 18

19 Unique, dual-matched indices mitigate index hopping during multiplexed target enrichment There are low levels of index hopping in multiplexed target enrichment Index hopping reads are filtered out using unique dual-matched indices 19

20 Index hopping reads are effectively filtered out with unique dual-matched indices Index hopping reads are filtered out with unique dual-matched adapters Reads would have been misassigned with combinatorial indices 20

21 Sources of sample cross-talk Index contamination Index hopping Index swapping Index cross-talk Spreading of signal 21

22 Index hopping during multiplexed sequencing Patterned flow cells utilize exclusion amplification (ExAmp) chemistry, associated with more index mis-assignment than bridge amplification Sinha R, Stanley G, et al. (2017) Index switching causes spreading-of-signal among multiplexed samples in Illumina HiSeq 4000 DNA sequencing. biorxiv. 22

23 Index hopping during multiplexed sequencing Index hopping can occur on the P5 and P7 side pdf?linkId=

24 Illumina s recommendations pdf?linkId=

25 Sources of sample cross-talk Index contamination Index hopping Index swapping Index cross-talk Spreading of signal Index mis-assignment Demultiplexing noise 25

26 Index design Errors can be introduced during synthesis, library preparation, amplification, and sequencing Need unique sequence tags for each sample Consider errors (insertions, deletions, and substitutions) Indices based on edit metric or Levenshtein distance are used to account for substitutions and indels Need to: avoid homopolymers, match GC content, exclude self-complements, color balance, and consider sequencing platform (4 vs. 2 color) Faircloth BC, Glenn TC. (2012) Not all sequence tags are created equal: Designing and validating sequence identification tags robust to indels. PLOS ONE, 7(8):e

27 Edit distance considers substitutions and indels Faircloth BC, Glenn TC. (2012) Not all sequence tags are created equal: Designing and validating sequence identification tags robust to indels. PLOS ONE, 7(8):e

28 Index quality filtering Quality filtering index reads minimizes cross-talk while preserving the majority of reads Unique, dual indexing is required for highly sensitive applications Run-specific and applicationspecific thresholds can be used to minimize cross-talk Wright ES, Vetsigian KH. (2016) Quality filtering of Illumina index reads mitigates sample cross-talk. BMC Genomics, 17(1):

29 Unique, dual-matched indices reduce sample cross-talk 29

30 Illumina and IDT partner on NGS multiplexing and exome enrichment The proprietary index kits will be compatible with Illumina library prep products and sequencers Highly optimized for use on platforms with Illumina s two-channel chemistry and patterned flow cells such as the NovaSeq Series Extend the number of unique dual indexes (UDI) from 8 UDIs to 24 The new 24 UDI kits can be preordered from Illumina now Future expansion to 96 UDI kits planned in partnership with Illumina The optimized index codes are now available through IDT for incorporation into custom adapter orders 30

31 xgen Dual Index UMI Adapter 3-in-1 design 31

32 Conclusions All sequencing platforms are susceptible to cross-talk for multiplexed sensitive applications Unique dual indices mitigate sample cross-talk and enable sensitive applications IDT has 384 predesigned unique indices for custom adapters Edit distance 3, color balanced as sets of 4, designed for 2- and 4-color sequencers, and with 50% GC content Contact Application Support for xgen Dual Index UMI Adapters or help with custom designs 32

33 Additional resources Kircher M, Sawyer S, et al. (2012) Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. Nucleic Acids Research, 40(1):e3 e3. D Amore R, Ijaz UZ, et al. (2016) A comprehensive benchmarking study of protocols and sequencing platforms for 16s rrna community profiling. BMC Genomics, 17(1):55. Sinha R, Stanley G, et al. (2017) Index switching causes spreading-of-signal among multiplexed samples in Illumina HiSeq 4000 DNA sequencing. biorxiv. Faircloth BC, Glenn TC. (2012) Not all sequence tags are created equal: Designing and validating sequence identification tags robust to indels. PLOS ONE, 7(8):e Wright ES, Vetsigian KH. (2016) Quality filtering of Illumina index reads mitigates sample crosstalk. BMC Genomics, 17(1):876. enseqlopedia.com/2016/12/index-mis-assignment-between-samples-on-hiseq-4000-and-x-ten/

34 THANK YOU 34