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CRISPR/Cas9-targeted removal of unwanted sequences from small-RNA sequencing libraries

Hardigan, Andrew A, Roberts, Brian S, Moore, Dianna E, Ramaker, Ryne C, Jones, Angela L, Myers, Richard M·2019·Nucleic Acids Research
DOI 10.1093/nar/gkz425PMC PMC6698666

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Abstract

In small RNA (smRNA) sequencing studies, highly abundant molecules such as adapter dimer products and tissue-specific microRNAs (miRNAs) inhibit accurate quantification of lowly expressed species. We previously developed a method to selectively deplete highly abundant miRNAs. However, this method does not deplete adapter dimer ligation products that, unless removed by gel-separation, comprise most of the library. Here, we have adapted and modified recently described methods for CRISPR/Cas9–based Depletion of Abundant Species by Hybridization (‘DASH’) to smRNA-seq, which we have termed miRNA and Adapter Dimer—DASH (MAD-DASH). In MAD-DASH, Cas9 is complexed with single guide RNAs (sgRNAs) targeting adapter dimer ligation products, alongside highly expressed tissue-specific smRNAs, for cleavage in vitro. This process dramatically reduces adapter dimer and targeted smRNA sequences, can be multiplexed, shows minimal off-target effects, improves the quantification of lowly expressed miRNAs from human plasma and tissue derived RNA, and obviates the need for gel-separation, greatly increasing sample throughput. Additionally, the method is fully customizable to other smRNA-seq preparation methods. Like depletion of ribosomal RNA for mRNA-seq and mitochondrial DNA for ATAC-seq, our method allows for greater proportional read-depth of non-targeted sequences.

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Figures

Figure 1. Depletion of adapter dimer and overabundant miRNAs with MAD-DASH. (A) MAD-DASH employs Cas9 and adapter dimer- or miRNA-specific sgRNAs to selectively deplete these sequences from final libraries. Cleaved sequences will not amplify during the second round of PCR amplification, and are also no longer suitable substrates for bridge amplification during Illumina sequencing (though they will still be able to bind to the flow-cell using the P5/P7 sequences remaining on each cleaved library). (B) Design of adapter dimer and hsa-miR-16–5p sgRNAs using available pre-existing PAM sites on our smRNA-seq 5′- and 3′-adapters. Adapter dimer targeting sgRNAs use a minus strand ‘NGG’ and have 10 bp of homology to both the 5′- and 3′ adapter, ensuring target cleavage specificity for adapter dimer sequences. hsa-miR-16–5p uses a 3′ PAM site one base away from the 3′—adapter ligation junction, which can be generalized to other smRNAs and provides highly specific targeting while minimizing off-targets. Shown are only the plus strand of non-PCR tailed sequences. Green boxes indicate the plus strand sequence corresponding to sgRNA location in the dsDNA library, while the yellow box indicates the plus strand location of the ‘NGG’ PAM in the dsDNA library. sgRNA location is depicted in blue along with the three 5′ ‘G’ nucleotides necessary for high levels of T7 in vitro transcription.
Figure 1. Depletion of adapter dimer and overabundant miRNAs with MAD-DASH. (A) MAD-DASH employs Cas9 and adapter dimer- or miRNA-specific sgRNAs to selectively deplete these sequences from final libraries. Cleaved sequences will not amplify during the second round of PCR amplification, and are also no longer suitable substrates for bridge amplification during Illumina sequencing (though they will still be able to bind to the flow-cell using the P5/P7 sequences remaining on each cleaved library). (B) Design of adapter dimer and hsa-miR-16–5p sgRNAs using available pre-existing PAM sites on our smRNA-seq 5′- and 3′-adapters. Adapter dimer targeting sgRNAs use a minus strand ‘NGG’ and have 10 bp of homology to both the 5′- and 3′ adapter, ensuring target cleavage specificity for adapter dimer sequences. hsa-miR-16–5p uses a 3′ PAM site one base away from the 3′—adapter ligation junction, which can be generalized to other smRNAs and provides highly specific targeting while minimizing off-targets. Shown are only the plus strand of non-PCR tailed sequences. Green boxes indicate the plus strand sequence corresponding to sgRNA location in the dsDNA library, while the yellow box indicates the plus strand location of the ‘NGG’ PAM in the dsDNA library. sgRNA location is depicted in blue along with the three 5′ ‘G’ nucleotides necessary for high levels of T7 in vitro transcription.CC BY-NC · PubMed Central
Figure 2. MAD-DASH smRNA-seq reduces adapter dimer and hsa-miR-16–5p in human brain total RNA samples. (A) Averaged read fraction of downsampled and CPM normalized adapter dimer sequences in gel extracted and bead cleanup samples. MAD-DASH in bead cleanup samples significantly depletes adapter dimer individually and in combination with depletion of hsa-miR-16–5p simultaneously. Asterisks indicate that a given read fraction was determined to be significantly altered compare to the respective cleanup's no MAD-DASH control using DESeq2 (FDR < 0.05). (B) Plot depicting the difference in the number of mature miRNA species reaching a specified count per million threshold between bead cleanup adapter dimer MAD-DASH smRNA-seq replicates and MAD-DASH control replicates. Downsampling and CPM normalization was performed as described in ‘Materials and Methods’ section. (C–F) Read counts from normalized replicate groups for bead cleanup treated versus control MAD-DASH samples targeting (C) adapter dimer (D) hsa-miR-16–5p and (E) adapter dimer and hsa-miR-16–5p (F) adapter dimer compared to gel extracted no-Cas9/sgRNA control. Significantly different sequences are filled black circles, with those having a log2-fold-change > 1 being labeled with text. Non-significantly different miRNAs are depicted as open gray circles. Adapter dimer and hsa-miR-16–5p are depicted as red circles with the same gray or black fill to denote significance. Significance was determined with DESeq2 and set as a Benjamini–Hochberg corrected P-value < 0.05.
Figure 2. MAD-DASH smRNA-seq reduces adapter dimer and hsa-miR-16–5p in human brain total RNA samples. (A) Averaged read fraction of downsampled and CPM normalized adapter dimer sequences in gel extracted and bead cleanup samples. MAD-DASH in bead cleanup samples significantly depletes adapter dimer individually and in combination with depletion of hsa-miR-16–5p simultaneously. Asterisks indicate that a given read fraction was determined to be significantly altered compare to the respective cleanup's no MAD-DASH control using DESeq2 (FDR < 0.05). (B) Plot depicting the difference in the number of mature miRNA species reaching a specified count per million threshold between bead cleanup adapter dimer MAD-DASH smRNA-seq replicates and MAD-DASH control replicates. Downsampling and CPM normalization was performed as described in ‘Materials and Methods’ section. (C–F) Read counts from normalized replicate groups for bead cleanup treated versus control MAD-DASH samples targeting (C) adapter dimer (D) hsa-miR-16–5p and (E) adapter dimer and hsa-miR-16–5p (F) adapter dimer compared to gel extracted no-Cas9/sgRNA control. Significantly different sequences are filled black circles, with those having a log2-fold-change > 1 being labeled with text. Non-significantly different miRNAs are depicted as open gray circles. Adapter dimer and hsa-miR-16–5p are depicted as red circles with the same gray or black fill to denote significance. Significance was determined with DESeq2 and set as a Benjamini–Hochberg corrected P-value < 0.05.CC BY-NC · PubMed Central
Figure 3. Rational design of a modified 5′ adapter with alternate PAM site enhances depletion of adapter dimer with MAD-DASH. (A) Design of the modified 5′ adapter compared to the multiplex 5′ adapter. Compared to the 10 bp competitor mismatch—10 bp competitor match—PAM design used in our first MAD-DASH iteration, the modified adapter uses 15 bp competitor mismatch—4 bp competitor match—PAM design and is predicted to have as much as 100-fold less binding to other non-target sequences in the library. Shown are the plus strands of non-PCR tailed sequences. Red boxes indicate the plus strand sequence corresponding to competitor match sequence in the dsDNA library, while the gray box indicates the sequence corresponding to sequence that drives adapter dimer target specificity, i.e. competitor mismatch sequence. The yellow box indicates the plus strand location of the ‘NGG’ PAM in the dsDNA library. sgRNA location is depicted in blue along with the three 5′ ‘G’ nucleotides necessary for high levels of T7 in vitro transcription. (B) MAD-DASH smRNA-seq using the modified 5′ adapter demonstrates substantially greater depletion of adapter dimer in bead cleanup samples than when using the multiplex adapter, and achieves a lower average read fraction than in modified adapter MAD-DASH control gel extraction samples. Normalized read fraction of downsampled and CPM normalized adapter dimer sequences for both adapter strategies are depicted. Reductions of adapter dimer in both gel extracted and bead cleanup samples were significant (DESeq2 adjusted P-value < 0.05). (C and D) Read counts from normalized replicate treated vs control groups prepared using the modified 5′ adapter. Bead cleanup MAD-DASH samples depleted of (C) modified adapter dimer and (D) bead cleanup modified adapter dimer compared to gel extracted no-Cas9/sgRNA modified adapter control are shown. Significantly different sequences are filled black circles, with those having a log2-fold-change > 1 being labeled with text. Non-significantly different miRNAs are depicted as open gray circles. Adapter dimer and hsa-miR-16–5p are depicted as red circles with the same grey or black fill to denote significance. Significance was determined with DESeq2 and set as a Benjamini–Hochberg corrected P-value < 0.05
Figure 3. Rational design of a modified 5′ adapter with alternate PAM site enhances depletion of adapter dimer with MAD-DASH. (A) Design of the modified 5′ adapter compared to the multiplex 5′ adapter. Compared to the 10 bp competitor mismatch—10 bp competitor match—PAM design used in our first MAD-DASH iteration, the modified adapter uses 15 bp competitor mismatch—4 bp competitor match—PAM design and is predicted to have as much as 100-fold less binding to other non-target sequences in the library. Shown are the plus strands of non-PCR tailed sequences. Red boxes indicate the plus strand sequence corresponding to competitor match sequence in the dsDNA library, while the gray box indicates the sequence corresponding to sequence that drives adapter dimer target specificity, i.e. competitor mismatch sequence. The yellow box indicates the plus strand location of the ‘NGG’ PAM in the dsDNA library. sgRNA location is depicted in blue along with the three 5′ ‘G’ nucleotides necessary for high levels of T7 in vitro transcription. (B) MAD-DASH smRNA-seq using the modified 5′ adapter demonstrates substantially greater depletion of adapter dimer in bead cleanup samples than when using the multiplex adapter, and achieves a lower average read fraction than in modified adapter MAD-DASH control gel extraction samples. Normalized read fraction of downsampled and CPM normalized adapter dimer sequences for both adapter strategies are depicted. Reductions of adapter dimer in both gel extracted and bead cleanup samples were significant (DESeq2 adjusted P-value < 0.05). (C and D) Read counts from normalized replicate treated vs control groups prepared using the modified 5′ adapter. Bead cleanup MAD-DASH samples depleted of (C) modified adapter dimer and (D) bead cleanup modified adapter dimer compared to gel extracted no-Cas9/sgRNA modified adapter control are shown. Significantly different sequences are filled black circles, with those having a log2-fold-change > 1 being labeled with text. Non-significantly different miRNAs are depicted as open gray circles. Adapter dimer and hsa-miR-16–5p are depicted as red circles with the same grey or black fill to denote significance. Significance was determined with DESeq2 and set as a Benjamini–Hochberg corrected P-value < 0.05CC BY-NC · PubMed Central
Figure 4. MAD-DASH smRNA-seq reduces adapter dimer and hsa-miR-16–5p in human plasma samples. (A) Adapter dimer concentrations in control or treated MAD-DASH smRNA-seq replicates (n = 3 in each group) made using the multiplex 5′ adapter (magenta) or the modified 5′ adapter (blue) were determined using qPCR and a synthetic adapter dimer library standard. Multiplex adapter sample concentration were adjusted 4× to account for the three other equimolar multiplex adapters. (B andC) Sequencing read counts from gel extracted, normalized replicate groups are plotted for treated vs control MAD-DASH samples using either (B) multiplex adapters and targeting hsa-miR-16–5p, or using (C) modified adapter and targeting adapter dimer. hsa-miR-16–5p depletion would not expected to vary between multiplex versus modified adapter use due to an identical sgRNA/PAM location. Adapter dimer depletion depicted in (C) represents a depletion of residual adapter dimer remaining after gel extraction, indicating that modified adapter dimer MAD-DASH can yield significant library improvements even when using traditional library cleanup methods. As in previous figures, significantly different sequences are filled black circles, with those having a log2-fold-change > 1 being labeled with text. Non-significantly different miRNAs are depicted as open gray circles. Adapter dimer and hsa-miR-16–5p are depicted as red circles with the same grey or black fill to denote significance. Significance was determined with DESeq2 and set as a Benjamini–Hochberg corrected P-value < 0.05. (D) Averaged read fraction of downsampled and CPM normalized adapter dimer sequences, hsa-miR-16–5p, non-hsa-miR-16–5p miRNAs and non-miRNA usable reads (see ‘Materials and Methods’ section) in modified adapter control miRNA band gel extracted (abbreviated as GE) samples, bead cleanup adapter dimer MAD-DASH samples and control and adapter dimer MAD-DASH smRNA region GE samples (E) Plot depicting the difference in the number of mature miRNA species reaching a specified count per million threshold between gel extracted or bead cleanup MAD-DASH plasma samples targeting adapter dimer. Downsampling and CPM normalization was performed as described in ‘Materials and Methods’ section. (F) Sequencing read counts from normalized replicate groups are plotted for bead cleanup adapter dimer MAD-DASH versus miRNA band gel control extracted samples. Significantly different sequences are filled black circles, with those having a log2-fold-change > 1 and a base mean of 100 being labeled with text to prevent overlapping labels of more lowly abundant significant sequences. Non-significantly different miRNAs are depicted as open gray circles. Adapter dimer and hsa-miR-16–5p are depicted as red circles with the same gray or black fill to denote significance. Significance was determined with DESeq2 and set as a Benjamini–Hochberg corrected P-value < 0.05.
Figure 4. MAD-DASH smRNA-seq reduces adapter dimer and hsa-miR-16–5p in human plasma samples. (A) Adapter dimer concentrations in control or treated MAD-DASH smRNA-seq replicates (n = 3 in each group) made using the multiplex 5′ adapter (magenta) or the modified 5′ adapter (blue) were determined using qPCR and a synthetic adapter dimer library standard. Multiplex adapter sample concentration were adjusted 4× to account for the three other equimolar multiplex adapters. (B andC) Sequencing read counts from gel extracted, normalized replicate groups are plotted for treated vs control MAD-DASH samples using either (B) multiplex adapters and targeting hsa-miR-16–5p, or using (C) modified adapter and targeting adapter dimer. hsa-miR-16–5p depletion would not expected to vary between multiplex versus modified adapter use due to an identical sgRNA/PAM location. Adapter dimer depletion depicted in (C) represents a depletion of residual adapter dimer remaining after gel extraction, indicating that modified adapter dimer MAD-DASH can yield significant library improvements even when using traditional library cleanup methods. As in previous figures, significantly different sequences are filled black circles, with those having a log2-fold-change > 1 being labeled with text. Non-significantly different miRNAs are depicted as open gray circles. Adapter dimer and hsa-miR-16–5p are depicted as red circles with the same grey or black fill to denote significance. Significance was determined with DESeq2 and set as a Benjamini–Hochberg corrected P-value < 0.05. (D) Averaged read fraction of downsampled and CPM normalized adapter dimer sequences, hsa-miR-16–5p, non-hsa-miR-16–5p miRNAs and non-miRNA usable reads (see ‘Materials and Methods’ section) in modified adapter control miRNA band gel extracted (abbreviated as GE) samples, bead cleanup adapter dimer MAD-DASH samples and control and adapter dimer MAD-DASH smRNA region GE samples (E) Plot depicting the difference in the number of mature miRNA species reaching a specified count per million threshold between gel extracted or bead cleanup MAD-DASH plasma samples targeting adapter dimer. Downsampling and CPM normalization was performed as described in ‘Materials and Methods’ section. (F) Sequencing read counts from normalized replicate groups are plotted for bead cleanup adapter dimer MAD-DASH versus miRNA band gel control extracted samples. Significantly different sequences are filled black circles, with those having a log2-fold-change > 1 and a base mean of 100 being labeled with text to prevent overlapping labels of more lowly abundant significant sequences. Non-significantly different miRNAs are depicted as open gray circles. Adapter dimer and hsa-miR-16–5p are depicted as red circles with the same gray or black fill to denote significance. Significance was determined with DESeq2 and set as a Benjamini–Hochberg corrected P-value < 0.05.CC BY-NC · PubMed Central
Figure 5. Illustration of MAD-DASH and standard smRNA-seq workflows. While MAD-DASH involves an extra PCR amplification in addition to the MAD-DASH CRISPR/Cas9 in vitro digestion, the extra ∼3 h to accomplish this relative to finishing the PCR in our standard protocol are more than offset by no longer needing to perform gel extraction, DNA recovery and library concentration. For nearly a hundred samples, we estimate that researcher time for smRNA-seq library construction can be reduced to only 1 day using MAD-DASH smRNA-seq and that this throughput can be further improved with greater protocol familiarity or automation.
Figure 5. Illustration of MAD-DASH and standard smRNA-seq workflows. While MAD-DASH involves an extra PCR amplification in addition to the MAD-DASH CRISPR/Cas9 in vitro digestion, the extra ∼3 h to accomplish this relative to finishing the PCR in our standard protocol are more than offset by no longer needing to perform gel extraction, DNA recovery and library concentration. For nearly a hundred samples, we estimate that researcher time for smRNA-seq library construction can be reduced to only 1 day using MAD-DASH smRNA-seq and that this throughput can be further improved with greater protocol familiarity or automation.CC BY-NC · PubMed Central

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Metadata

Authors
Hardigan, A. A., Roberts, B. S., Moore, D. E., Ramaker, R. C., Jones, A. L., & Myers, R. M.
Affiliations
HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806, USA
ORCID
Hardigan, Andrew A
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2019-06-05
Volume
47
Issue
14
Pages
e84-e84
ISSN
  • 0305-1048
  • 1362-4962
DOI
10.1093/nar/gkz425
PMC
PMC6698666
Cited by
37
References
7
Language
en
Licence
creativecommons.org/licenses/by-nc/4.0/ (vor)
Funders
  • University of Alabama at Birmingham (TR0001417)
  • UAB Medical Scientist Training Program (5T32GM008361)

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