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Maximizing the acquisition of unique reads in noninvasive capture sequencing experiments
Molecular Ecology Resources ( IF 7.7 ) Pub Date : 2020-11-20 , DOI: 10.1111/1755-0998.13300 Claudia Fontsere 1 , Marina Alvarez-Estape 1 , Jack Lester 2 , Mimi Arandjelovic 2 , Martin Kuhlwilm 1 , Paula Dieguez 2 , Anthony Agbor 2 , Samuel Angedakin 2 , Emmanuel Ayuk Ayimisin 2 , Mattia Bessone 2 , Gregory Brazzola 2 , Tobias Deschner 2 , Manasseh Eno-Nku 3 , Anne-Céline Granjon 2 , Josephine Head 2 , Parag Kadam 4 , Ammie K Kalan 2 , Mohamed Kambi 2 , Kevin Langergraber 5, 6 , Juan Lapuente 2, 7 , Giovanna Maretti 2 , Lucy Jayne Ormsby 2 , Alex Piel 8 , Martha M Robbins 2 , Fiona Stewart 4, 8 , Virginie Vergnes 9 , Roman M Wittig 2, 10 , Hjalmar S Kühl 2, 11 , Tomas Marques-Bonet 1, 12, 13, 14 , David A Hughes 15, 16 , Esther Lizano 1, 14
Molecular Ecology Resources ( IF 7.7 ) Pub Date : 2020-11-20 , DOI: 10.1111/1755-0998.13300 Claudia Fontsere 1 , Marina Alvarez-Estape 1 , Jack Lester 2 , Mimi Arandjelovic 2 , Martin Kuhlwilm 1 , Paula Dieguez 2 , Anthony Agbor 2 , Samuel Angedakin 2 , Emmanuel Ayuk Ayimisin 2 , Mattia Bessone 2 , Gregory Brazzola 2 , Tobias Deschner 2 , Manasseh Eno-Nku 3 , Anne-Céline Granjon 2 , Josephine Head 2 , Parag Kadam 4 , Ammie K Kalan 2 , Mohamed Kambi 2 , Kevin Langergraber 5, 6 , Juan Lapuente 2, 7 , Giovanna Maretti 2 , Lucy Jayne Ormsby 2 , Alex Piel 8 , Martha M Robbins 2 , Fiona Stewart 4, 8 , Virginie Vergnes 9 , Roman M Wittig 2, 10 , Hjalmar S Kühl 2, 11 , Tomas Marques-Bonet 1, 12, 13, 14 , David A Hughes 15, 16 , Esther Lizano 1, 14
Affiliation
Noninvasive samples as a source of DNA are gaining interest in genomic studies of endangered species. However, their complex nature and low endogenous DNA content hamper the recovery of good quality data. Target capture has become a productive method to enrich the endogenous fraction of noninvasive samples, such as faeces, but its sensitivity has not yet been extensively studied. Coping with faecal samples with an endogenous DNA content below 1% is a common problem when prior selection of samples from a large collection is not possible. However, samples classified as unfavourable for target capture sequencing might be the only representatives of unique specific geographical locations, or to answer the question of interest. To explore how library complexity may be increased without repeating DNA extractions and generating new libraries, in this study we captured the exome of 60 chimpanzees (Pan troglodytes) using faecal samples with very low proportions of endogenous content (<1%). Our results indicate that by performing additional hybridizations of the same libraries, the molecular complexity can be maintained to achieve higher coverage. Also, whenever possible, the starting DNA material for capture should be increased. Finally, we specifically calculated the sequencing effort needed to avoid exhausting the library complexity of enriched faecal samples with low endogenous DNA content. This study provides guidelines, schemes and tools for laboratories facing the challenges of working with noninvasive samples containing extremely low amounts of endogenous DNA.
中文翻译:
在无创捕获测序实验中最大限度地获取独特的读数
作为 DNA 来源的非侵入性样本越来越受到濒危物种基因组研究的关注。然而,它们的复杂性和低内源性 DNA 含量阻碍了高质量数据的恢复。目标捕获已成为一种富集无创样品(如粪便)内源性成分的有效方法,但其敏感性尚未得到广泛研究。当无法从大量样本中预先选择样本时,处理内源性 DNA 含量低于 1% 的粪便样本是一个常见问题。然而,归类为不利于目标捕获测序的样本可能是独特特定地理位置的唯一代表,或者回答感兴趣的问题。为了探索如何在不重复 DNA 提取和生成新文库的情况下增加文库复杂性,Pan troglodytes ) 使用内源性含量比例非常低 (<1%) 的粪便样本。我们的结果表明,通过对相同文库进行额外的杂交,可以保持分子复杂性以实现更高的覆盖率。此外,只要有可能,应增加用于捕获的起始 DNA 材料。最后,我们专门计算了避免耗尽内源性 DNA 含量低的富集粪便样本的文库复杂性所需的测序工作。这项研究为面临处理内源性 DNA 含量极低的非侵入性样本挑战的实验室提供了指导方针、计划和工具。
更新日期:2020-11-20
中文翻译:
在无创捕获测序实验中最大限度地获取独特的读数
作为 DNA 来源的非侵入性样本越来越受到濒危物种基因组研究的关注。然而,它们的复杂性和低内源性 DNA 含量阻碍了高质量数据的恢复。目标捕获已成为一种富集无创样品(如粪便)内源性成分的有效方法,但其敏感性尚未得到广泛研究。当无法从大量样本中预先选择样本时,处理内源性 DNA 含量低于 1% 的粪便样本是一个常见问题。然而,归类为不利于目标捕获测序的样本可能是独特特定地理位置的唯一代表,或者回答感兴趣的问题。为了探索如何在不重复 DNA 提取和生成新文库的情况下增加文库复杂性,Pan troglodytes ) 使用内源性含量比例非常低 (<1%) 的粪便样本。我们的结果表明,通过对相同文库进行额外的杂交,可以保持分子复杂性以实现更高的覆盖率。此外,只要有可能,应增加用于捕获的起始 DNA 材料。最后,我们专门计算了避免耗尽内源性 DNA 含量低的富集粪便样本的文库复杂性所需的测序工作。这项研究为面临处理内源性 DNA 含量极低的非侵入性样本挑战的实验室提供了指导方针、计划和工具。