Whole-Genome Sequence of an Indian Group A Streptococcus emm Type 1-2 Strain Isolated from a Blood Sample in North India.

Group A Streptococcus emm type 1-2 is more prevalent than emm type 1 in India. Only partial information is available about the genetic characteristics of this type. Here, genome sequencing of emm type 1-2 strain 1085 (from blood) was conducted. A contig 2,010,300 bp long, with a total of 1,877 annotated proteins, was obtained (NCBI accession number CP047120, assembly accession number ASM983284v1).

ANNOUNCEMENT

Group A Streptococcus (GAS) (Streptococcus pyogenes), which is responsible for many life-threatening invasive infections, is a highly diverse and rapidly emerging pathogen (1, 2). As more than 200 emm types of GAS have been identified worldwide, attempts to develop a vaccine have failed (3). Even emm-typing data from India are different from those of other countries (3–6). Our previous data showed that emm1, which is prevalent worldwide, is rare in India. Instead, emm1-2 strains are more common in India (7). Type emm1-2 strains have been isolated from throat, skin, and also blood samples, indicating its invasive nature. Our reports based on PCR and microarray analysis have explored little about this type (5, 7). So, whole-genome sequencing of a type emm1-2 strain was carried out here.

Our study was approved by the Institute Ethics Committee (IEC) of Post Graduate Institute of Medical Education & Research (PGIMER) in Chandigarh, India (INT/IEC/2018/000760). The emm1-2 strain (1085) was isolated from a blood sample of a patient suffering from septicemia who was hospitalized at PGIMER (3, 8). The strain was isolated using a blood culturing technique as per the standardized protocol. For genomic DNA isolation, Todd-Hewitt broth with 0.2% yeast extract was used. Culturing was done at 37°C. Genomic DNA was isolated using zirconium beads in combination with a DNeasy kit (Qiagen, Germany). A hybrid assembly was generated from Illumina (Genome Analyzer IIx; RTA version 1.8.70.0) and Nanopore (GridION-X5) data. Libraries of 320 bp were prepared according to Illumina’s instructions, “Preparing Samples for Paired-End-Sequencing.” For Nanopore data, DNA from the sample was end repaired using the NEBNext Ultra II end repair kit (catalog number E7546L; New England BioLabs, USA) and cleaned up with 1× AmPure beads (Beckmann Coulter, USA). Native barcode ligation was performed with a blunt/TA (T4 DNA) ligase mix (M0367L; New England BioLabs) using the native barcoding genomic DNA kit (EXP-NBD104; Oxford Nanopore Technologies, UK) and cleaned with 1× AmPure beads. Qubit-quantified barcode-ligated DNA sample libraries were pooled at equimolar concentrations to attain a 600-ng pooled sample. Adapter ligation (BAM) was performed for 10 min using the NEBNext quick ligation module (E6056L; New England BioLabs). The library mix was cleaned up using 0.6× AmPure beads, and finally, the sequencing library was eluted in 15 μl of elution buffer. Sequencing was performed on a SpotON flow cell R9.4 (Oxford Nanopore Technologies). For quality checking, the minimum threshold quality score for Nanopore data was a Q-score of 7. Raw data were also error corrected using Illumina data and assembled using Maryland Super Read Cabog Assembler (MaSuRCA) version 3.3 (9). The gene and protein predictions were carried out using Prokka version 1.14 (10). The predicted proteins were used for a similarity search using the Diamond version 0.8.29 BLASTP program (11). All software was used with default parameters.

Approximately 194,811 reads were generated. The number of reads by Illumina sequencing was 10,184,236, with an absolute length of 2 × 110 bp. For Nanopore sequencing, the total read length was 350,972,921, with an average of 1,801.6 bp and an N50 value of 2,807 bp. The assembly resulted in a contig 2,010,300 bp long, which showed homology with GAS. The G+C content of 38.4% was comparable with that in earlier reports (12). Out of a total of 2,055 genes, 1,937 were complementary determining sequences (CDSs) and 1,877 were annotated (Fig. 1). A total of 118 genes coding for different RNAs were also identified, of which 8 each encoded 5S, 16S, and 23S rRNAs.

FIG 1

Gene ontology (GO) association of predicted protein-coding genes of the emm1-2 GAS strain 1085.

Data availability.

This information has been deposited at DDBJ/EMBL/GenBank under the accession number CP047120. The raw data were submitted to the Sequence Read Archive (SRA) with the accession number PRJNA596618.