Этот пример показывает, как считать функции из парного секвенирования после выравнивания их по всему геному человека, курированному консорциумом Genome Reference. Этот пример использует Genome Ссылки Consortium Human Build 38 закрашенных фигур релиза 12 (GRCh38.p12) в качестве ссылки генома человека.
Этот пример работает только на платформах UNIX ® и Mac. Загрузите пакет поддержки Bioinformatics Toolbox™ Interface for Bowtie Aligner из Add-On Explorer. Для получения дополнительной информации смотрите Пакеты поддержки ПО Bioinformatics Toolbox.
Этот пример предполагает, что у вас есть:
Загружена и извлечена сборка RefSeq из Genome Ссылки Consortium Human Build 38 закрашенных фигур релиза 12 (GRCh38.p12).
Загружены и организованы некоторые парные считывания данных. Этот пример использует данные секвенирования экзома из проекта 1000 геномов. Парные чтения обозначаются '_ 1' и '_ 2' в именах файлов.
Создайте индекс для выравнивания показаний по ссылке с помощью bowtie2build. Файл GCF_000001405.38_GRCh38.p12_genomic.fna содержит человеческий эталонный геном в формате FASTA. bowtieIdx - базовое имя файлов ссылки индекса. The '--threads 8' Опция определяет количество параллельных потоков для быстрого создания индекса файлов. Вам не нужно указывать полные пути к файлам * .fna или * .index, если пример запускается из того же расположения папки. Укажите полные пути, если вы хотите хранить файлы в другом месте или запустить пример из другой папки.
bowtieIdx = 'GCF_000001405.38_GRCh38.p12_genomic.index'; buildFlag = bowtie2build('GCF_000001405.38_GRCh38.p12_genomic.fna',... bowtieIdx,'--threads 8');
Выравнивание показаний парного конца по ссылке с помощью bowtie2. Можно создать Bowtie2AlignOptions объект для задания различных опций, таких как количество параллельных потоков для использования.
opt = Bowtie2AlignOptions; opt.NumThreads = 8; reads1 = 'HG00096_1.fastq'; reads2 = 'HG00096_2.fastq'; bowtie2(bowtieIdx,reads1,reads2,'HG00096.sam',opt);
Файлы SAM могут быть очень большими. Использование BioMap чтобы выбрать только данные для правильной ссылки. В данном примере рассмотрим APOE, который является геном хромосомы 19, связанным с болезнью Альцгеймера. Создайте меньший файл BAM для APOE, чтобы улучшить эффективность.
apoeRef = 'NC_000019.10'; % Reference name for Chromosome 19 in HG38 bm = BioMap('HG00096.sam','SelectReference',apoeRef); write(bm, 'HG00096.bam','Format','bam');
Warning: Found invalid tag in header type: 'PG'. Ignoring tag 'PN:bowtie2'. Warning: The read sequences in input SAM file do not appear to be ordered according to the start position of their alignments with the reference sequence. Because of this, there will be a decrease in performance when accessing the reads. For maximum performance, order the read sequences in the SAM file, before creating a BioMap object.
Использование featurecount сравнить количество транскриптов для каждого варианта APOE с использованием GTF- файла. Полная таблица функций включена в сборку GRCh38.p12 в формате GFF, которая может быть преобразована в GTF с помощью cuffgffread. Этот пример использует упрощенный GTF на основе транскриптов APOE. APOE_gene.gtf входит в комплект программного обеспечения.
[FeatTable, Summary] = featurecount('APOE_gene.gtf','HG00096.bam',... 'Metafeature','transcript_id');
Processing GTF file APOE_gene.gtf ... Processing BAM file HG00096.bam ... Processing reference NC_000019.10 ... 10000 reads processed ... 20000 reads processed ... 30000 reads processed ... 40000 reads processed ... 50000 reads processed ... 60000 reads processed ... 70000 reads processed ... 80000 reads processed ... 90000 reads processed ... 100000 reads processed ... 110000 reads processed ... 120000 reads processed ... 130000 reads processed ... 140000 reads processed ... 150000 reads processed ... 160000 reads processed ... 170000 reads processed ... 180000 reads processed ... 190000 reads processed ... 200000 reads processed ... 210000 reads processed ... 220000 reads processed ... 230000 reads processed ... 240000 reads processed ... 250000 reads processed ... 260000 reads processed ... 270000 reads processed ... 280000 reads processed ... 290000 reads processed ... 300000 reads processed ... 310000 reads processed ... 320000 reads processed ... 330000 reads processed ... 340000 reads processed ... 350000 reads processed ... 360000 reads processed ... 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Done.
bamsort | BioMap | bowtie2 | bowtie2build | bwamem | cuffgffread | cufflinks | featurecount | samsort