Этот пример показывает, как считать функции от чтений упорядочивания парного конца после выравнивания их к целому геному человека курировавшими Консорциумом Ссылки Генома. Этот пример использует Консорциальную Сборку Человека Ссылки Генома 38 релизов 12 (GRCh38.p12) закрашенной фигуры в качестве ссылки генома человека.
Этот пример работает над UNIX® и платформами Mac только. Загрузите Интерфейс Bioinformatics Toolbox™ для пакета поддержки Выравнивателя Галстука-бабочки из Add-On Explorer.
Этот пример принимает, что вы имеете:
Загруженный и извлеченный блок RefSeq от Консорциальной Сборки Человека Ссылки Генома 38 релизов 12 (GRCh38.p12) закрашенной фигуры.
Загруженный и организованный некоторый парный конец считывает данные. Этот пример использует exome упорядочивание данных из этих 1 000 проектов геномов. Чтения парного конца обозначаются '_1' и '_2' в именах файлов после инвентарного номера. Вот одна возможность для того, как данные могут быть организованы в папках:
sequence/ +--HG00096/ | +--SRR077487_1.filt.fastq | +--SRR077487_2.filt.fastq | +--SRR081241_1.filt.fastq | +--SRR081241_2.filt.fastq | +--HG00097 +-- SRR765989_1.filt.fastq +-- SRR765989_2.filt.fastq
Создайте индекс для выравнивания чтений к ссылке с помощью bowtie2build. Файл GCF_000001405.38_GRCh38.p12_genomic.fna содержит человеческий ссылочный геном в формате FASTA. bowtieIdx является базовым именем файлов справочного указателя. Опция '--threads 8' задает количество параллельных потоков, чтобы создать индексные файлы быстрее.
bowtieIdx = 'GCF_000001405.38_GRCh38.p12_genomic.index'; buildFlag = bowtie2build('GCF_000001405.38_GRCh38.p12_genomic.fna',... bowtieIdx,'--threads 8');
Используйте функцию помощника, которую alignAllReads, чтобы выровнять каждый набор парного конца читает в ссылку. Функция производит файл SAM в текущей папке для каждой выборки в папке 'последовательности'.
addpath(fullfile(matlabroot,'examples','bioinfo','main')); % Make sure the supporting files are on the search path type alignAllReads
function samFiles = alignAllReads(indexBaseName, inputDir, outputDir)
%ALIGNALLREADS Helper function for CountFeaturesExample
%
% SAMFILES = alignAllReads(INDEXBASENAME, INPUTDIR, OUTPUTDIR) aligns
% paired-end sequencing reads using Bowtie2 to a prebuilt index,
% producing SAM-formatted alignment files. INDEXBASENAME
% is a character vector specifying the prefix of the index files
% created with BOWTIE2BUILD. INPUTDIR contains subdirectories for each
% sample containing paired-end reads in FASTQ format:
%
% INPUTDIR/
% +-- HG00096/
% | +-- SRR077487_1.filt.fastq
% | +-- SRR077487_2.filt.fastq
% | +-- SRR081241_1.filt.fastq
% | +-- SRR081241_2.filt.fastq
% |
% +-- HG00097/
% +-- SRR765989_1.filt.fastq
% +-- SRR765989_2.filt.fastq
%
% NOTE: each mate is distinguished by the '_1' or '_2' after the
% accession number in the filename.
%
% SAMFILES is a cell array of the resulting SAM-formatted files created
% with Bowtie2, and are placed in OUTPUTDIR.
% Copyright 2018 The MathWorks, Inc.
% Use dir() to identify sample names (subfolders of inputDir).
d = dir(inputDir);
samples = {d(3:end).name}; % skip special '.' & '..' folders
samFiles = strcat(samples, '.sam');
for s=1:numel(samples)
% Identify mate pairs of reads for each sample
sampleReadsPath = fullfile(inputDir, samples{s});
reads1 = dir([sampleReadsPath '/*_1*']);
reads2 = dir([sampleReadsPath '/*_2*']);
reads1 = fullfile(sampleReadsPath, {reads1(:).name});
reads2 = fullfile(sampleReadsPath, {reads2(:).name});
% Get full filename to SAM file
samFiles{s} = fullfile(outputDir, samFiles{s});
% Perform alignment, if file doesn't exist
if ~exist(samFiles{s}, 'file')
bowtie2(indexBaseName, reads1, reads2, samFiles{s}, '-p 2');
end
end
end
samFiles = alignAllReads(bowtieIdx,'sequence','.');
Файлы SAM могут быть очень большими. Используйте BioMap, чтобы выбрать только данные для правильной ссылки. В данном примере рассмотрите APOE, который является геном на Хромосоме 19 соединенных к болезни Альцгеймера. Создайте набор меньших файлов BAM для APOE, чтобы улучшать производительность.
apoeRef = 'NC_000019.10'; % Reference name for Chromosome 19 in HG38 bamFiles = strrep(samFiles,'.sam','.bam'); for i=1:numel(samFiles) if ~exist(bamFiles{i},'file') bm = BioMap(samFiles{i},'SelectReference',apoeRef); write(bm, bamFiles{i},'Format','bam'); end end
Используйте featurecount, чтобы сравнить количество расшифровок стенограммы для каждого варианта APOE с помощью файла GTF. Полная таблица функций включена в блок GRCh38.p12 в формате GFF, который может быть преобразован в GTF с помощью cuffgffread. Этот пример использует упрощенный GTF на основе расшифровок стенограммы APOE. APOE_gene.gtf включен с программным обеспечением.
[FeatTable, Summary] = featurecount('APOE_gene.gtf',bamFiles,... '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.
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