Trinucleotide Repeats

To construct a sgRNA expression vector, protospacer sequences should not contain repeat sequence as follows: more than 4 continuous T nucleotides (4∼6 nucleotide poly (T) tract acts as a termination signal for RNA pol III), or other homopolymer sequences (more than 5 continuous A or C or G, more than 6 dinucleotide or trinucleotide repeats). This step can be performed by check_sgRNA_seq.pl. Once candidate CRISPR target sites are determined, selected sequences can be used to design oligonucleotides. As described above, the sequence pattern of CRISPR target sites found by sgRNAcas9.pl are 5′-GGX18NGG-3′, 5′-GX19NGG-3′ or 5′-X20NGG-3′. Therefore, the sequence of GGX18, GX19 or X20 will be extracted and used directly to design 20-nt length of sgRNAs by using sgRPrimer.pl. To describe how to use this script to batch design oligonucleotides for constructing sgRNA expression vector, the pGL3-U6-gRNA-Puromycin vector (modified from Addgene 51133) was selected as an example, which is designed for expressing customizable sgRNA under control of the U6 promoter. Annealed oligos were cloned into the vector at a Bsa I restriction site. To facilitate cloning of the 20 bp target sequence, extra bases need to be added to the ends. In this study, ‘accg’ was added to the 5′ end of the sense oligo and ‘aaac’ to the 5′ end of reverse complementary sequence (anti-sense oligo). Then, equal amounts of the sense and anti-sense strands were synthesized and annealed to generate the ds-oligo. This product can be easily ligated into the digested pGL3-U6-gRNA-Puromycin vector.
To investigate on- or off-target cleavage effects, certain lengths of predicted sequence need to be extracted from the genome by nucleotide positions. Then cleavage sites can be validated by using the T7 endonucleases I (T7E1) assay or sequencing. This is another time-consuming step. To raise experiment efficiency and save time, extraction of target sequence by nucleotide position can be performed by extract_targetSeq.pl. The length of sequences extracted from genome was set as an optional argument in this program. A default parameter value was provided to extract DNA fragments up to 1,000 bp in length. Then the sequence was used as a template to design PCR primer pairs for validation of the Cas9 cleavage effect.

For a given TR unit, the associated repeat type is defined as follows: All TRs with units that differ from the given repeat unit only by circular permutations and/or the reverse complement are associated to the same repeat type. Clearly, there are always several repeat units, which belong to the same repeat type. We follow the convention to represent a repeat type by that unit which comes first in an alphabetical ordering of all units that are associated to it [54 (link)]. This convention allows us to count and identify repeat units without reference to the repeat unit phase or strand. To give an example, the repeat type represented by the unit AAG incorporates all TRs with units AAG, AGA, GAA, TTC, TCT, and CTT. Furthermore, the term repeat motif is used instead of the term repeat type when we aim at distinguishing between sense and anti-sense strand repeat characteristics, but not the repeat phase. Hence, on the level of repeat motifs, AAG, AGA, GAA are all represented by AAG, but are distinguished from the repeat motif CTT, which also represents TTC and TCT. Finally, the terms repeat type and repeat motif are distinguished from the term repeat class which we use to denote the collection of all repeats with the same repeat unit size (e.g. mono-, di-, trinucleotide repeats).
An important property of one or a set of TR types is their density within a nucleotide sequence. It is defined as the fraction of base pairs that are found within repeats of a given set of repeat types over the total number of base pairs in the sequence. Repeat type densities are measured in base pairs per megabase pairs (bp/Mbp). It can be envisaged as the coverage of the sequence with the specified repeat types. Since in several genomes, including D. pulex, the number of (Ns) contributes significantly to the total size, all TR densities computed in this work were corrected for the number of Ns. It is important to distinguish repeat densities from densities based on number counts of repeats (measured in counts/Mbp) that are sometimes used in publications, e.g. [44 (link),47 (link),51 (link)].

The aim was to describe and analyse trends in the Cryptosporidium species and gp60 genotypes identified in human outbreaks of cryptosporidiosis in England and Wales from January 2009 to December 2017. Definitions used to define an outbreak were: an incident in which two or more people experienced a similar illness and linked in time or place, or a greater than expected rate of Cryptosporidium reports compared with the usual background rate for a place and time. Cryptosporidium outbreaks were extracted from the eFOSS database and from records for those that also came to the attention of the national CRU during outbreak investigations. The proportions of outbreak routes of transmission were compared between the two databases by uncorrected Chi square and a P-value of 0.05 was regarded as significant. The databases were reconciled by PHE centre, setting/place name, postcode, dates of first and last known cases, and populated with Cryptosporidium species and gp60 subtypes identified in the stools of cases and any additional samples tested. The outbreaks were analysed for trends in vehicles and settings, season, and associated Cryptosporidium species and gp60 subtypes. The CRU archive and the NCBI nucleotide DB and PubMed were searched for previous reports of subtypes found.
To identify species, Cryptosporidium positive stools were sent by primary diagnostic laboratories to the national CRU, generally within 5 days of collection [13 (link)]. Oocysts were separated from faecal material by salt flotation, disrupted by boiling, and DNA extracted using proteinase K digestion and a spin column kit (QIAamp DNA mini kit, Qiagen, Hilden, Germany) as described previously [13 (link)]. Samples were screened for C. parvum and C. hominis using a duplex real-time PCR assay [46 (link)] and other species were sought using a nested PCR targeting the SSU rDNA gene [47 (link)]. A nested PCR targeting the gp60 gene was used to subtype C. parvum and C. hominis samples known or suspected to be part of outbreaks as described previously [48 (link)]; to simplify workflow a cocktail of single round PCR primers was developed and used from 2015, as described previously [49 (link)]. PCR amplicons were subjected to bidirectional sequencing (Applied Biosystems 3500XL) and sequence similarities searched for in the NCBI Blastn website tools. Gp60 subtypes were confirmed by manual identification of trinucleotide repeats and other repeat sequences (Fig. 1). The findings were contextualised at the time to inform outbreak investigations and updated for this article.
In animal contact outbreaks, animals were sampled by a Veterinary Investigation Officer if requested by the outbreak control team and tested using immunofluorescence microscopy (Crypto-cel, Cellabs) at the Animal and Plant Health Agency’s central laboratory, Weybridge. Cryptosporidium-positive samples were sent to the CRU for genotyping as described above. In recreational and drinking water outbreaks, sampling and testing was undertaken as described in [7 ] if requested by the outbreak control team. Cryptosporidium positive microscope slides sent to the CRU for genotyping were processed as described previously [37 (link)] until 2015. After 2015 DNA extraction from slides was done using a chelex-based method as described previously [50 ].

Family member BI underwent comprehensive genetic assessment. A panel for trinucleotide repeats for spinocerebellar atrophies was performed at MNG laboratories. Exome sequencing was also performed as a duo, with the index mother (A2) at Invitae. Whole-genome sequencing was performed as a trio with the index mother (A2) and the 29-year-old unaffected sister (BVI) at UCLA’s California Center for Rare Diseases as previously described.^{15 (link)} Targeted sequencing was performed on family members BII and BVI at the UCLA Orphan Disease Testing Center.

Microsatellite sequences from O. vulgare L. ssp. hirtum were identified as a service by Ecogenics GmbH following NGS sequencing of an O. vulgare L. ssp. hirtum genomic DNA sample. The Illumina TruSeq nano DNA library was sequenced on an Illumina MiSeq sequencing platform using a nano v2 500 cycles sequencing chip. The resulting paired-end reads which passed Illumina’s chastity filter were subjected to de-multiplexing and trimming of Illumina adaptor residuals. Subsequently the quality of the surviving reads was checked with FastQC v0.11.8 [14 ]. In a next step, the paired-end reads were quality filtered and merged with USEARCH v11.0.667 [15 (link)] to in silico reform the sequenced molecules. The resulting merged reads were screened with the software Tandem Repeats Finder, v4.09 [16 (link)]. After this process, 6121 merged reads contained a microsatellite insert with a tetra- or a trinucleotide of at least six repeat units or a dinucleotide of at least ten repeat units. Primer design was performed with primer 3 [17 (link),18 (link)]. Raw NGS sequences can be accessed at the NCBI Sequence Read Archive under project number PRJNA921701.