Illumina Figure showing a paired-end flow cell for MiSeq, HiSeq 2000/2500 and NovaSeq 6000
Sample Multiplexing Sample multiplexing allows large numbers of libraries to be pooled and sequenced on a single run. This will drastically reduce cost. There are 384 Unique Dual Indexes (Sets A, B, C and D). This means a total of 384 samples can be multiplexed on a single lane.
Adapter Trimming Adapter trimming is the process of removing adapter sequences from the 3’ ends of reads. Adapter sequences should be removed from reads because they interfere with downstream analyses, such as alignment of reads to a reference. it is necessary to eliminate adapter sequences from reads. These adapter sequences contain important elements, including the sequencing primer binding sites, the index sequences, and the sites that facilitate the attachment of library fragments to the flow cell lawn. You can use trimmomatic for this trimming step. Some assemblers give you the option to trim the reads as part of the assembly process.
Genome Assembly In bioinformatics, genome assembly is the process of putting a large number of short DNA sequences into the correct order to recreate the original genome from which the DNA came. There are two types of genome assembly: de novo assembly and mapping/reference-based assembly.
- The process starts with a reads file (sample.fastq). Sequence reads can be submitted to databases like Sequence Reads Archive (SRA).
- De novo assembly is the process of creating a novel genome from scratch without the aid of reference/template data. This is the standard approach for bacteria. The most common assembly mechanism for short reads is de Bruijn graph, which is a directed graph that represents overlaps between sequences of symbols.
- A reference/mapping-based assembly needs a reference genome to be used as a representative example of a species’ set of genes. Once the reference genome is available, the genome assembly of every new sample becomes much easier and quicker. This is the standard approach for human and viruses genomes.
- The final product is usually a fasta file that has segments of contiguous base pairs (1 or more contig/s).
- Draft genome: Multiple contigs per chromosome or genomic interspersed with gaps of unknown sequences.
- Complete genome: 1 single contig for the entire chromosome/plasmid.
- We usually submit genome assemblies to databases such as European Nucleotide Archive and NCBI Assembly (GenBank).
What do make the assembler task more difficult?
- Contamination
- Adapter Sequences not trimmed
- Repetitive Sequences
- Low Coverage
- Poor Quality Reads
Assembly Statistics
| Metric | Description |
|---|---|
| N50 | Sequence length of shortest contig at 50% of the assembly length |
| L50 | The smallest number of conitgs whose bases sum makes 50% of of the assembly length |
| Coverage | Number of Seq reads covering each nucleotide in the genome (e.g. 40x) |
It is a tool to assemble bacterial isolate genomes from Illumina paired-end reads. The SPAdes genome assembler is the standard de novo genome assembler for Illumina whole genome sequencing data of bacteria and other small microbes. Shovill is a pipeline which uses SPAdes at its core, but it is faster.
If you get an error saying Cannot open temporary file kmc_00253.bin and Could not determine genome size from '', use ulimit -n 2048 to increase limit for opened files for the kmc tool on MacOS.
Let's assemble our first genome. To follow the steps here, you need to download the files available here.
conda activate shovill
shovill --trim --assembler skesa --outdir out_S56 --R1 S56_R1.fastq --R2 S56_R2.fastq
mash screen -w -p 8 ../../problem_set1/RefSeqSketches.msh contigs.fa > S56_screen_winning_contigs.tab
sort -gr S56_screen_winning_contigs.tab > S56_screen_winning_contigs_sorted.tab
less S56_screen_winning_contigs_sorted.tab | head -n 10
QUAST stands for QUality ASsessment Tool. The tool evaluates genome assemblies by computing various metrics. It has an interactive visualizer for the outputs.
Let's try it! Go to NCBI and download the reference genome and gff file for Pseudomonas aeruginosa (PAO1).
conda activate quast
quast.py contigs.fa -r GCF_000006765.1_ASM676v1_genomic.fna.gz
It is a tool to assess the genome assembly and annotation completeness based on evolutionarily informed expectations of gene content (single-copy orthologs).
conda activate busco
busco -i ~/MGJW/problem_set3/out_S56/contigs.fa -l bacteria_odb10 -o busco_op -m genome
busco -i ~/MGJW/problem_set1/fasta/genome4.fasta -l bacteria_odb10 -o busco_op2 -m genome
busco -i ~/MGJW/problem_set1/fasta/genome4.fasta -l clostridiales_odb10 -o busco_op3 -m genome