reverse_complement()
- 对序列取反补
seq = 'ATCG'
rc_seq = reverse_complement(seq)
translate()
- 将DNA序列翻译成氨基酸序列
dna_seq = 'ATGCTGAACTG'
aa_seq = translate(dna_seq)
gc_content()
- 计算序列的GC含量
seq = 'ATCGATCG'
gc = gc_content(seq)
subsample()
- 对序列进行子抽样
input = fastq('input.fastq.gz')
subsampled = subsample(input, fraction=0.1)
merge()
- 合并两个或多个序列文件
input1 = fastq('input1.fastq.gz')
input2 = fastq('input2.fastq.gz')
merged = merge(input1, input2)
average_quality()
- 计算序列的平均质量
input = fastq('input.fastq.gz')
avg_qual = average_quality(input)
trim_polya()
- 对序列进行poly(A)尾修剪
input = fastq('input.fastq.gz')
trimmed = trim_polya(input)
annotate()
- 对序列进行注释
input = ... # 一些序列
annotation = ... # 一些注释信息
annotated = annotate(input, with=annotation)
to_fasta()
- 将序列文件转换为FASTA格式
input = fastq('input.fastq.gz')
fasta = to_fasta(input)
to_fastq()
- 将序列文件转换为FASTQ格式
input = fasta('input.fasta')
fastq = to_fastq(input)
reverse()
- 对序列进行反转
seq = 'ATCG'
reversed_seq = reverse(seq)
complement()
- 对序列进行互补
seq = 'ATCG'
comp_seq = complement(seq)
is_paired()
- 判断序列是否成对出现
input = fastq('input.fastq.gz')
paired = is_paired(input)
pair()
- 对成对的序列进行配对
input = fastq('input.fastq.gz')
paired = pair(input)
is_unique()
- 判断序列是否唯一
input = fastq('input.fastq.gz')
unique = is_unique(input)
unique_only()
- 选择唯一的序列
input = fastq('input.fastq.gz')
unique = unique_only(input)
random()
- 生成一个随机数
rand_num = random()
shuffle()
- 对序列进行随机重排
input = fastq('input.fastq.gz')
shuffled = shuffle(input)
annotate_gff()
- 对序列进行基因组注释
input = fasta('input.fasta')
gff_file = 'annotation.gff'
annotated = annotate_gff(input, gff_file)
align()
- 对序列进行局部或全局比对
input = fasta('input.fasta')
ref_seq = fasta('reference.fasta')
alignment = align(input, ref_seq)
align_sam()
- 对序列进行SAM格式比对
input = fasta('input.fasta')
sam_file = 'alignment.sam'
aligned = align_sam(input, sam_file)
align_bam()
- 对序列进行BAM格式比对
input = fasta('input.fasta')
bam_file = 'alignment.bam'
aligned = align_bam(input, bam_file)
compress()
- 对文件进行压缩
input_file = 'input.txt'
compressed_file = compress(input_file, format='gzip')
decompress()
- 对文件进行解压缩
compressed_file = 'input.gz'
decompressed_file = decompress(compressed_file)
distance()
- 计算两个序列之间的距离
seq1 = 'ATCG'
seq2 = 'AGCG'
dist = distance(seq1, seq2)
intersect()
- 计算两个序列集合的交集
input1 = fasta('input1.fasta')
input2 = fasta('input2.fasta')
intersected = intersect(input1, input2)
union()
- 计算两个序列集合的并集
input1 = fasta('input1.fasta')
input2 = fasta('input2.fasta')
unioned = union(input1, input2)
subtract()
- 计算两个序列集合的差集
input1 = fasta('input1.fasta')
input2 = fasta('input2.fasta')
subtracted = subtract(input1, input2)
average()
- 计算一组数字的平均值
data = [1, 2, 3, 4, 5]
avg = average(data)
maximum()
- 计算一组数字的最大值
data = [1, 2, 3, 4, 5]
max_val = maximum(data)
minimum()
- 计算一组数字的最小值
data = [1, 2, 3, 4, 5]
min_val = minimum(data)
standard_deviation()
- 计算一组数字的标准差
data
常见生信分析代码片段
下面是常用的NGless功能函数处理宏基因组数据的代码片段:
从FASTQ文件中过滤低质量的reads,并将结果输出到新文件中:
ngless "1.0"
input = fastq('input.fq')
preprocess(input, phred=33) using |read|:
if read.avg_qual < 20:
discard
input | keep | add_sequence_length | sum | write(`filtered.fq`, compression=Fastq)
根据OTU表将reads映射到参考数据库,并生成OTU表:
ngless "1.0"
input = fastq('input.fq')
reference = fasta('ref.fasta')
mapped = map(input, reference, exact=False, sensitive=True)
otu_table(mapped, reference) | write(`otu_table.txt`, format="csv")
对OTU表进行物种注释,并生成注释表:
ngless "1.0"
otu_table = csv('otu_table.csv')
annotation_db = csv('annotation_db.csv')
annotated = annotate_species(otu_table, annotation_db)
annotated | write(`annotated_otu_table.csv`, format="csv")
根据OTU丰度信息生成热图:
ngless "1.0"
otu_table = csv('otu_table.csv')
heatmap(otu_table) | write(`heatmap.png`, format="png")
对样品进行稀释,并生成稀释后的OTU表:
ngless "1.0"
otu_table = csv('otu_table.csv')
diluted = dilute(otu_table, factor=10)
diluted | write(`diluted_otu_table.csv`, format="csv")
对OTU表进行组间差异分析,使用差异显著性检验方法:
ngless "1.0"
otu_table = csv('otu_table.csv')
groups = csv('groups.csv')
differential(abundance(otu_table), groups) | write(`differential_analysis.csv`, format="csv")
对样品进行Beta多样性分析,并生成PCoA图:
ngless "1.0"
otu_table = csv('otu_table.csv')
pcoa_table = pcoa(otu_table)
pcoa_table | plot(`pcoa.png`, format="png")
对OTU表进行功能注释,并生成功能注释表:
ngless "1.0"
otu_table = csv('otu_table.csv')
gene_db = csv('gene_db.csv')
annotated = annotate_functions(otu_table, gene_db)
annotated | write(`annotated_otu_table.csv`, format="csv")
根据OTU表进行物种多样性分析,并生成物种多样性指数表:
ngless "1.0"
otu_table = csv('otu_table.csv')
diversity_indices(otu_table) | write(`diversity_indices.csv`, format="csv")
对样品进行Alpha多样性分析,并生成稀释曲线图:
ngless "1.0"
otu_table = csv('otu_table.csv')
alpha_table = alpha_diversity(otu_table)
alpha_table | plot(`alpha_diversity.png`, format="png")
对OTU表进行物种丰度分析,并生成物种丰度柱状图:
ngless "1.0"
otu_table = csv('otu_table.csv')
species_abundance(otu_table) | plot(`species_abundance.png`, format="png")
根据OTU表进行共生网络分析,并生成共生网络图:
ngless "1.0"
otu_table = csv('otu_table.csv')
cooccurrence_network(otu_table) | plot(`cooccurrence_network.png`, format="png")
对样品进行微生物组成分析,并生成样品组成饼图:
ngless "1.0"
otu_table = csv('otu_table.csv')
sample_composition(otu_table) | plot(`sample_composition.png`, format="png")
对OTU表进行代谢通路分析,并生成代谢通路富集柱状图:
ngless "1.0"
otu_table = csv('otu_table.csv')
pathway_db = csv('pathway_db.csv')
enriched_pathways(otu_table, pathway_db) | plot(`enriched_pathways.png`, format="png")
对OTU表进行进化分析,并生成进化树:
ngless "1.0"
otu_table = csv('otu_table.csv')
evolutionary_analysis(otu_table) | plot(`evolutionary_tree.png`, format="png")
将多个OTU表进行合并:
ngless "1.0"
otu_table1 = csv('otu_table1.csv')
otu_table2 = csv('otu_table2.csv')
combined = merge(otu_table1, otu_table2)
combined | write(`merged_otu_table.csv`, format="csv")
对OTU表进行样品分类,并生成分类树:
ngless "1.0"
otu_table = csv('otu_table.csv')
taxonomy_tree(otu_table) | plot(`taxonomy_tree.png`, format="png")
根据OTU表进行功能富集分析,并生成功能富集柱状图:
ngless "1.0"
otu_table = csv('otu_table.csv')
gene_set_db = csv('gene_set_db.csv')
enriched_functions(otu_table, gene_set_db) | plot(`enriched_functions.png`, format="png")
对样品进行Gamma多样性分析,并生成Gamma多样性曲线:
ngless "1.0"
otu_table = csv('otu_table.csv')
gamma_table = gamma_diversity(otu_table)
gamma_table | plot(`gamma_diversity.png`, format="png")
对OTU表进行进化树构建,并生成进化树:
ngless "1.0"
otu_table = csv('otu_table.csv')
phylogenetic_tree(otu_table) | plot(`phylogenetic_tree.png`, format="png")
请注意,这些代码片段仅为示例,并不一定适用于所有NGless版本和数据集。在实际使用时,请根据具体情况进行修改和调整。
NGLess的使用示例
人类肠道宏基因组学的功能和分类分析
步骤概述:
- 准备数据:获取并准备用于分析的原始测序数据。
- 质量控制和过滤:对原始数据进行质量控制、去除低质量序列和去除宿主DNA等。
- 人类肠道宏基因组分类:对数据进行分类,识别宿主肠道微生物。
- 功能注释:对宏基因组数据进行功能注释,识别和分析肠道微生物的功能特征。
详细步骤:
步骤 1: 准备数据
- 下载并准备用于肠道宏基因组学的原始测序数据,例如来自人类肠道样本的元组装数据(metagenomic sequencing data)。
步骤 2: 质量控制和过滤
- 使用 NGLess 进行质量控制和过滤,例如使用 FastQC 进行质量评估,然后使用 NGLess 进行质量过滤:
-
ngless 'input = fastq(‘data.fastq.gz’)
preprocess(input, keep_singles=False) using |read|:
if len(read) < 45:
discard;
elif count(N) > 0.1:
discard;
else:
keep;
##### 步骤 3: 人类肠道宏基因组分类
* 使用 NGLess 和相应的数据库进行宏基因组分类。示例中使用 Silva 数据库进行分类:
ngless ‘input = fastq(‘filtered_data.fastq.gz’)
preprocessed = preprocess(input)
m1 = map(preprocessed, reference=‘silva’)’
##### 步骤 4: 功能注释
* 对宏基因组数据进行功能注释,例如使用 Diamond 或者 BLAST 进行序列比对,并使用相应的数据库(例如KEGG、COG、GO等)进行功能注释:
ngless ‘input = fastq(‘filtered_data.fastq.gz’)
preprocessed = preprocess(input)
m1 = map(preprocessed, reference=‘silva’)
annotated = annotate(m1, data=‘kegg’)’
#### 注意事项:
* 这些是一些基本步骤,具体的分析流程和参数设置可能会因实验设计和所使用的数据库而有所不同。
* 示例代码中使用了 NGLess 的 DSL 语法,实际使用时可能需要根据具体的数据和数据库进行调整和优化。
* 在执行代码之前,请确保您已了解数据处理和分析的需求,并正确设置参数以获得最佳结果。
#### 全流程脚本
ngless “1.0”
import “parallel” version “0.6”
import “mocat” version “0.0”
import “motus” version “0.1”
import “igc” version “0.0”
samples = readlines(‘igc.demo.short’)
sample = lock1(samples)
input = load_fastq_directory(sample)
input = preprocess(input, keep_singles=False) using |read|:
read = substrim(read, min_quality=25)
if len(read) < 45:
discard
mapped = map(input, reference=‘hg19’)
mapped = select(mapped) using |mr|:
mr = mr.filter(min_match_size=45, min_identity_pc=90, action={unmatch})
if mr.flag({mapped}):
discard
input = as_reads(mapped)
mapped = map(input, reference=‘igc’, mode_all=True)
counts = count(mapped,
features=[‘KEGG_ko’, ‘eggNOG_OG’],
normalization={scaled})
collect(counts,
current=sample,
allneeded=samples,
ofile=‘igc.profiles.txt’)
mapped = map(input, reference=‘motus’, mode_all=True)
counted = count(mapped, features=[‘gene’], multiple={dist1})
motus_table = motus(counted)
collect(motus_table,
current=sample,
allneeded=samples,
ofile=‘motus-counts.txt’)
## 案例分析脚本demo
[https://ngless.embl.de/\_static/gut-metagenomics-tutorial-presentation/scripts/1\_preproc.ngl]( )
ngless “0.0”
import “mocat” version “0.0”
input = load_mocat_sample(‘SAMN05615097.short’)
preprocess(input, keep_singles=False) using |read|:
read = substrim(read, min_quality=25)
if len(read) < 45:
discard
write(input, ofile=‘preproc.fq.gz’)
[https://ngless.embl.de/\_static/gut-metagenomics-tutorial-presentation/scripts/2\_map\_hg19.ngl]( )
ngless “0.0”
import “mocat” version “0.0”
input = load_mocat_sample(‘SAMN05615097.short’)
preprocess(input, keep_singles=False) using |read|:
read = substrim(read, min_quality=25)
if len(read) < 45:
discard
mapped = map(input, reference=‘hg19’)
mapped = select(mapped) using |mr|:
mr = mr.filter(min_match_size=45, min_identity_pc=90, action={unmatch})
if mr.flag({mapped}):
discard
write(mapped, ofile=‘mapped.bam’)
[https://ngless.embl.de/\_static/gut-metagenomics-tutorial-presentation/scripts/3\_taxprofile.ngl]( )
ngless “0.0”
import “mocat” version “0.0”
import “motus” version “0.1”
import “specI” version “0.1”
input = load_mocat_sample(‘SAMN05615097.short’)
preprocess(input, keep_singles=False) using |read|:
read = substrim(read, min_quality=25)
if len(read) < 45:
discard
mapped = map(input, reference=‘hg19’)
mapped = select(mapped) using |mr|:
mr = mr.filter(min_match_size=45, min_identity_pc=90, action={unmatch})
if mr.flag({mapped}):
discard
input = as_reads(mapped)
mapped = map(input, reference=‘motus’, mode_all=True)
mapped = select(mapped) using |mr|:
mr = mr.filter(min_match_size=45, min_identity_pc=97, action={drop})
if not mr.flag({mapped}):
discard
counted = count(mapped, features=[‘gene’], multiple={dist1})
write(motus(counted),
ofile=‘motus.counts.txt’)
input = as_reads(mapped)
mapped = map(input, reference=‘refmg’)
mapped = select(mapped) using |mr|:
mr = mr.filter(min_match_size=45, min_identity_pc=97, action={drop})
if not mr.flag({mapped}):
discard
write(count(mapped,
features=[‘species’],
include_minus1=True),
ofile=‘species.raw.counts.txt’)
[https://ngless.embl.de/\_static/gut-metagenomics-tutorial-presentation/scripts/4\_parallel.ngl]( )
ngless “0.0”
import “parallel” version “0.0”
import “mocat” version “0.0”
import “motus” version “0.1”
import “specI” version “0.1”
samples = readlines(‘igc.demo.short’)
sample = lock1(samples)
input = load_mocat_sample(sample)
preprocess(input, keep_singles=False) using |read|:
read = substrim(read, min_quality=25)
if len(read) < 45:
discard
mapped = map(input, reference=‘hg19’)
mapped = select(mapped) using |mr|:
mr = mr.filter(min_match_size=45, min_identity_pc=90, action={unmatch})
if mr.flag({mapped}):
discard
input = as_reads(mapped)
mapped = map(input, reference=‘motus’, mode_all=True)
counted = count(mapped, features=[‘gene’], multiple={dist1})
motus_table = motus(counted)
collect(motus_table,
current=sample,
allneeded=samples,
ofile=‘motus-counts.txt’)
input = as_reads(mapped)
mapped = map(input, reference=‘refmg’)
mapped = select(mapped) using |mr|:
mr = mr.filter(min_match_size=45, min_identity_pc=97, action={drop})
if not mr.flag({mapped}):
discard
collect(count(mapped,
features=[‘species’],
include_minus1=True),
current=sample,
allneeded=samples,
ofile=‘species.raw.counts.txt’)
[https://ngless.embl.de/\_static/gut-metagenomics-tutorial-presentation/scripts/5\_w\_igc.ngl]( )
ngless “0.0”
import “parallel” version “0.0”
import “mocat” version “0.0”
import “motus” version “0.1”
import “igc” version “0.0”
import “specI” version “0.1”
samples = readlines(‘igc.demo.short’)
sample = lock1(samples)
input = load_mocat_sample(sample)
preprocess(input, keep_singles=False) using |read|:
read = substrim(read, min_quality=25)
if len(read) < 45:
discard
mapped = map(input, reference=‘hg19’)
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