Here, Synthetic Biology Platform Lead at the Wyss Institute for Biologically Inspired Engineering, Richie Kohman, continues exploring the breakthroughs of Next Generation Sequencing (NGS)
Exploring the locations and identities of RNA within biological tissues can uncover important information about cell types and states as well as give insight into cellular development and diseased states. Until recently, it was not possible to probe these samples in a spatially defined way. Classic techniques such as bulk RNA sequencing homogenise tissues and eliminate any record of molecular location.
Several approaches have been developed that uncover this spatial record by repositioning the site of analysis away from the original tissue. For example, tissues can be micro-dissected to restrict data to a smaller region of interest, or RNA can be pulled from tissues and captured onto well-characterised surfaces. The focus of this piece, however, will be on a panel of techniques that analyse molecules directly within the tissue sample itself. These fully in situ approaches enable the best spatial understanding of targets because they are retained in their original three-dimensional position.(1)
The focus here will be on examples that use Next Generation Sequencing (NGS) as a readout. Other approaches, such as those that use fluorescent in situ hybridisation, will not be covered. Although not the focus here, there is one example where NGS was performed for in situ protein detection through the sequencing of DNA conjugated to antibodies.(2)
In general, fully in situ protocols can be broken into three steps:
- Library preparation.
In the library preparation step, the tissue is prepared for sequencing by performing a variety of biochemical steps that capture targets of interest and convert them into DNA amplicons. This amplification is necessary to increase the signal obtained from each molecule. In the sequencing step, chemistry is performed to read out nucleic acid sequences. These techniques are fluorescence-based with different spectral colours used to indicate which base is being identified. Iteration of the sequencing chemistry is used to uncover the string of bases.
In the analysis step, image stacks are processed to align the data and extract the positions and sequences of each captured target. Because the sequencing and analysis steps are more modular, the focus here will be on the differences in library preparations between techniques.
Methods will be categorised by whether they investigate a panel of RNA targets, or whether they aim to capture all RNA.
Targeted NGS of RNA within tissues is appealing if one has a defined number of transcripts to investigate. One of the earliest targeted examples came from a technique referred generally as In Situ Sequencing (ISS).(3) Here mRNA targets of interest are converted into complementary DNA (cDNA) sequences through a process called reverse transcription (RT). Targets are specified by using RT primers selective to the RNAs of interest. DNA amplicons are then created through the addition of DNA strands called padlock probes, which bind cDNA to great circular structures which can undergo a process called rolling circle amplification (RCA).
Although an effective strategy, the inefficiency of the RT step led researchers to investigate alternative approaches. Several groups(4, 5, 6), including ours(5, 6), reported direct DNA amplification off of RNA using padlock probes plus a specific ligase. BOLARAMIS(6) and targeted ExSeq(5) utilise SplintR ligase, whereas HybRISS (4) uses a proprietary ligase mixture. These methods report an increase in transcript detection compared to cDNA padlocking, highlighting the advantage of circumventing the reverse transcription step. Recently a technique called STARmap was reported that utilised pairs of DNA strands called snail probes during their sample preparation.(7) Snail probes consist of two strands that bind next to one another in an orientation where one probe acts as a splint to circularise the other. After ligation, RCA can produce sequence-able amplicons. These probes enable target-specific amplification because both probes must bind properly to produce an amplicon.
Untargeted in situ tissue NGS promises to produce the most complete and least biased depiction of a cell’s gene expression, however it is also the most challenging to perform. Fluorescent in situ sequencing (FISSEQ) was the first example of untargeted labelling of transcripts in situ.(8, 9) This technique utilised random RT primers containing a known attached sequence. After reverse transcription, cDNA circularisation was directly performed allowing RCA to be accomplished. More recently FISSEQ was combined with Expansion Microscopy to create a super-resolution version of the technique that was effective at de-crowding amplicon density and refining subcellular transcript locations. (5) Lastly, InstaSeq refined the reverse transcription step to favour mRNA detection over ribosomal RNA.
Despite being a relatively new field, spatial transcriptomics if progressing rapidly. In the years to come, expect further breakthroughs and the emergence of commercial ventures that more effectively disseminate the techniques to the scientific community.
- Turczyk, B. M. et al. Spatial Sequencing: A Perspective. J Biomol Tech 2020, 31 (2), 44-46.
- Kohman, R. E. and Church, G.M. Fluorescent in situ Sequencing of DNA Barcoded Antibodies. bioRxiv 2020, doi: doi.org/10.1101/2020.04.27.060624.
- Ke, R. et al. In situ Sequencing for RNA Analysis in Preserved Tissue and Cells. Nat Methods 2013, 10 (9), 857-860.
- Lee, H. et al. Direct RNA Targeted Transcriptomic Profiling in Tissue using Hybridization-based RNA In Situ Sequencing (HybRISS). bioRxiv 2020, doi: doi.org/10.1101/2020.12.02.408781.
- Alon, S. et al. Expansion Sequencing: Spatially Precise in situ Transcriptomics in Intact Biological Systems. Science 2021, 371, (6528), eaax2656 DOI: 10.1126/science.aax2656.
- Liu, S. et al. Barcoded Oligonucleotides Ligated on RNA Amplified for Multiplex and Parallel in-situ Analyses. Nucleic Acids Res 2021, 49 (10), gkab120, doi.org/10.1093/nar/gkab120.
- Wang, X. et al. Three-dimensional Intact-tissue Sequencing of Single-cell Transcriptional States. Science 2018, 361, (6400), eaat5691 DOI: 10.1126/science.aat5691.
- Lee, J. H. et al. Highly multiplexed subcellular RNA sequencing in situ. Science 2014, 343 (6177), 1360-1363.
- Lee, J. H. et al. Fluorescent in situ Sequencing (FISSEQ) of RNA for Gene Expression Profiling in Intact Cells and Tissues. Nat Protoc 2015, 10 (3), 442-458.
- Furth, D. et al. In Situ Transcriptome Accessibility Sequencing (INSTA-seq). bioRxiv 2019, doi: doi.org/10.1101/722819.
Please note: This is a commercial profile
Next Generation Sequencing Within Biological Tissues – Richie Kohman at the Wyss Institute
Richie Kohman, Synthetic Biology Platform Lead at the Wyss Institute for Biologically Inspired Engineering discusses his work on Next Generation Sequencing within biological tissues The Synthetic Biology Platform at the Wyss Institute is innovating the use of Next Generation Sequencing (NGS) to study intact biological tissues. NGS is one of the most advanced methods in […]
Editor’s Recommended Articles
Read more here: Source link