CRISPR-based targeted haplotype-resolved assemblies of a megabase region

Li, Taotao; Du, Duo; Zhang, Dandan; Ma, Jiakang; Zhou, Mengyu; Meng, Wen Jun; Jin, Zelin; Lin, Yu-Min; Chen, Ziqiang; Yuan, Hao; Wang, Jue; Dong, Sujun; Sun, Song; Ye, Wenjing; Li, Boshen; Zhang, Zhao; Xie, Zhi; Wan-hua, Qiu; Liu, Yun

doi:10.1101/2022.01.21.477044

Cited by 2 publications

(2 citation statements)

References 47 publications

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“…We modified the CaBagE method 33,34 and designed a tiling strategy of spacing gRNAs as previously shown 37 . Five gRNAs were designed to isolate four ~20 kb adjacent targets (T1-T4) from the human MSH2 locus.…”

Section: Target Excision and Isolation With A Modified Cabage Methodsmentioning

confidence: 99%

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Targeted Phasing of 2-200 Kilobase DNA Fragments with a Short-Read Sequencer and a Single-Tube Linked-Read Library Method

Mikhaylova¹,

Rzepka²,

Kawamura³

et al. 2023

Preprint

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In the human genome, heterozygous sites are genomic positions with different alleles inherited from each parent. On average, there is a heterozygous site every 1-2 kilobases (kb). Resolving whether two alleles in neighboring heterozygous positions are physically linked—that is, phased—is possible with a short-read sequencer if the sequencing library captures long-range information. TELL-Seq is a library preparation method based on millions of barcoded micro-sized beads that enables instrument-free phasing of a whole human genome in a single PCR tube. TELL-Seq incorporates a unique molecular identifier (barcode) to the short reads generated from the same high-molecular-weight (HMW) DNA fragment (known as ‘linked-reads’). However, genome-scale TELL-Seq is not cost-effective for applications focusing on a single locus or a few loci. Here, we present an optimized TELL-Seq protocol that enables the cost-effective phasing of enriched loci (targets) of varying sizes, purity levels, and heterozygosity. Targeted TELL-Seq maximizes linked-read efficiency and library yield while minimizing input requirements, fragment collisions on microbeads, and sequencing burden. To validate the targeted protocol, we phased seven 180-200 kb loci enriched by CRISPR/Cas9-mediated excision coupled with pulse-field electrophoresis, four 20 kb loci enriched by CRISPR/Cas9-mediated protection from exonuclease digestion, and six 2-13 kb loci amplified by PCR. The selected targets have clinical and research relevance (BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, PMS2, SCN5A-SCN10A, andPKI3CA). These analyses reveal that targeted TELL-Seq provides a reliable way of phasing allelic variants within targets (2-200 kb in length) with the low cost and high accuracy of short-read sequencing.

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Section: Target Excision and Isolation With A Modified Cabage Methodsmentioning

confidence: 99%

“…Such gRNA distribution would also enable the recovery of ~40 kb, ~60 kb, and ~80 kb fragments if fragments of these sizes were preserved during DNA extraction (Fig. 2A) 37 .…”

Section: Phasing Partially Enriched Mid-size Targetsmentioning

confidence: 99%

Targeted Phasing of 2-200 Kilobase DNA Fragments with a Short-Read Sequencer and a Single-Tube Linked-Read Library Method

Mikhaylova¹,

Rzepka²,

Kawamura³

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

Targeted phasing of 2–200 kilobase DNA fragments with a short-read sequencer and a single-tube linked-read library method

Mikhaylova,

Rzepka,

Kawamura

et al. 2024

Sci Rep

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In the human genome, heterozygous sites refer to genomic positions with a different allele or nucleotide variant on the maternal and paternal chromosomes. Resolving these allelic differences by chromosomal copy, also known as phasing, is achievable on a short-read sequencer when using a library preparation method that captures long-range genomic information. TELL-Seq is a library preparation that captures long-range genomic information with the aid of molecular identifiers (barcodes). The same barcode is used to tag the reads derived from the same long DNA fragment within a range of up to 200 kilobases (kb), generating linked-reads. This strategy can be used to phase an entire genome. Here, we introduce a TELL-Seq protocol developed for targeted applications, enabling the phasing of enriched loci of varying sizes, purity levels, and heterozygosity. To validate this protocol, we phased 2–200 kb loci enriched with different methods: CRISPR/Cas9-mediated excision coupled with pulse-field electrophoresis for the longest fragments, CRISPR/Cas9-mediated protection from exonuclease digestion for mid-size fragments, and long PCR for the shortest fragments. All selected loci have known clinical relevance: BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, PMS2, SCN5A-SCN10A, and PKI3CA. Collectively, the analyses show that TELL-Seq can accurately phase 2–200 kb targets using a short-read sequencer.

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CRISPR-based targeted haplotype-resolved assemblies of a megabase region

Cited by 2 publications

References 47 publications

Targeted Phasing of 2-200 Kilobase DNA Fragments with a Short-Read Sequencer and a Single-Tube Linked-Read Library Method

Targeted Phasing of 2-200 Kilobase DNA Fragments with a Short-Read Sequencer and a Single-Tube Linked-Read Library Method

Targeted phasing of 2–200 kilobase DNA fragments with a short-read sequencer and a single-tube linked-read library method

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